Methods and compositions for preventing or treating ophthalmic conditions

ABSTRACT

The disclosure generally describes methods of preventing or treating ophthalmic diseases or conditions in a mammalian subject, such as diabetic retinopathy, cataracts, retinitis pigmentosa, glaucoma, macular degeneration, choroidal neovascularization, retinal degeneration, and oxygen-induced retinopathy. The methods comprise administering an effective amount of an aromatic-cationic peptide to subjects in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/401,527, filed Jan. 9, 2017, now U.S. Pat. No. 10,188,692, which is acontinuation of U.S. application Ser. No. 14/671,538, filed Mar. 27,2015, now U.S. Pat. No. 9,549,963, which is a continuation of U.S.application Ser. No. 13/897,070, filed May 17, 2013, now U.S. Pat. No.9,023,807, which is a continuation of U.S. application Ser. No.12/861,593, filed Aug. 23, 2010, now U.S. Pat. No. 8,470,784, whichclaims priority to U.S. Provisional Application No. 61/236,440, filedAug. 24, 2009; U.S. Provisional Application No. 61/237,745, filed Aug.28, 2009; and U.S. Provisional Application No. 61/348,470, filed May 26,2010. The entire contents of these applications are hereby incorporatedby reference in their entireties.

TECHNICAL FIELD

The present technology relates generally to compositions and methods ofpreventing or treating ophthalmic diseases or conditions. In particular,the present technology relates to administering aromatic-cationicpeptides in effective amounts to prevent or treat ophthalmic diseases orconditions, e.g., diabetic retinopathy, cataracts, retinitis pigmentosa,glaucoma, choroidal neovascularization, and oxygen-induced retinopathy,in mammalian subjects.

BACKGROUND

The following description is provided to assist the understanding of thereader. None of the information provided or references cited is admittedto be prior art to the present invention.

Diseases and degenerative conditions of the optic nerve and retina arethe leading causes of blindness in the world. A significant degenerativecondition of the retina is age-related macular degeneration (ARMD). ARMDis the most common cause of blindness in people over 50 in the USA andits prevalence increases with age. ARMD is classified as either wet(neovascular) or dry (non-neovascular); the dry form of the disease ismore common. Macular degeneration occurs when the central retina hasbecome distorted and thinned usually associated with age but alsocharacterized by intra-ocular inflammation and angiogenesis (wet ARMDonly) and/or intra-ocular infection. The subsequent generation of freeradicals, resulting in oxidative tissue damage, local inflammation andproduction of growth factors (such as VEGF and FGF) and inflammatorymediators, leads to inappropriate neovascularisation in common with thewet form of ARMD.

Retinopathy is a leading cause of blindness in type I diabetes, and isalso common in type II diabetes. The degree of retinopathy depends onthe duration of diabetes, and generally begins to occur ten or moreyears after onset of diabetes. Diabetic retinopathy may be classified asnon-proliferative, where the retinopathy is characterized by increasedcapillary permeability, edema and exudates, or proliferative, where theretinopathy is characterized by neovascularisation extending from theretina to the vitreous, scarring, deposit of fibrous tissue and thepotential for retinal detachment. Diabetic retinopathy is believed to becaused by the development of glycosylated proteins due to high bloodglucose. Several other less common retinopathies include choroidalneovascular membrane (CNVM), cystoid macular edema (CME), epi-retinalmembrane (ERM) and macular hole.

Glaucoma is made up of a collection of eye diseases that cause visionloss by damage to the optic nerve. Elevated intraocular pressure (IOP)due to inadequate ocular drainage is the primary cause of glaucoma.Glaucoma often develops as the eye ages, or it can occur as the resultof an eye injury, inflammation, tumor or in advanced cases of cataractor diabetes. It can also be caused by the increase in IOP caused bytreatment with steroids. Drug therapies that are proven to be effectivein glaucoma reduce IOP either by decreasing vitreous humor production orby facilitating ocular draining. Such agents are often vasodilators andas such act on the sympathetic nervous system and include adrenergicantagonists.

SUMMARY

The present technology relates generally to the treatment or preventionof ophthalmic diseases or conditions in mammals through administrationof therapeutically effective amounts of aromatic-cationic peptides tosubjects in need thereof.

In one aspect, the present disclosure provides a method of treating orpreventing an ophthalmic condition in a mammalian subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of the peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or Phe-D-Arg-Phe-Lys-NH₂. In one embodiment,the ophthalmic condition is selected from the group consisting of:diabetic retinopathy, cataracts, retinitis pigmentosa, glaucoma, maculardegeneration, choroidal neovascularization, retinal degeneration, andoxygen-induced retinopathy.

In one aspect, the disclosure provides a method of treating orpreventing ophthalmic conditions in a mammalian subject, comprisingadministering to said mammalian subject a therapeutically effectiveamount of an aromatic-cationic peptide. In some embodiments, thearomatic-cationic peptide is a peptide having:

at least one net positive charge;

a minimum of four amino acids;

a maximum of about twenty amino acids;

a relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) wherein 3p_(m)is the largest number that is less than or equal to r+1; and arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1, except that when a is 1,p_(t) may also be 1. In particular embodiments, the mammalian subject isa human.

In one embodiment, 2p_(m) is the largest number that is less than orequal to r+1, and may be equal to p_(t). The aromatic-cationic peptidemay be a water-soluble peptide having a minimum of two or a minimum ofthree positive charges.

In one embodiment, the peptide comprises one or more non-naturallyoccurring amino acids, for example, one or more D-amino acids. In someembodiments, the C-terminal carboxyl group of the amino acid at theC-terminus is amidated. In certain embodiments, the peptide has aminimum of four amino acids. The peptide may have a maximum of about 6,a maximum of about 9, or a maximum of about 12 amino acids.

In one embodiment, the peptide may have the formulaPhe-D-Arg-Phe-Lys-NH₂ (SS-20) or 2′,6′-Dmp-D-Arg-Phe-Lys-NH₂. In aparticular embodiment, the aromatic-cationic peptide has the formulaD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (referred to interchangeably as SS-31,MTP-131, or Bendavia™).

In one embodiment, the peptide is defined by formula I:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independentlyselected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and n is an integer from 1 to 5.

In a particular embodiment, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰,R¹¹, and R¹² are all hydrogen; and n is 4. In another embodiment, R¹,R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹¹ are all hydrogen; R⁸ and R¹² aremethyl; R¹⁰ is hydroxyl; and n is 4.

In one embodiment, the peptide is defined by formula II:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

R³ and R⁴ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo;

R⁵, R⁶, R⁷, R⁸, and R⁹ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and n is an integer from 1 to 5.

The aromatic-cationic peptides may be administered in a variety of ways.In some embodiments, the peptides may be administered intraocularly,orally, topically, intranasally, intravenously, subcutaneously, ortransdermally (e.g., by iontophoresis).

In one aspect, the present disclosure provides a pharmaceuticalcomposition comprising a therapeutically effective amount of the peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or Phe-D-Arg-Phe-Lys-NH₂ formulated fortopical, iontophoretic, or intraocular administration.

In one aspect, the present disclosure provides an ophthalmic formulationcomprising a therapeutically effective amount of the peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or Phe-D-Arg-Phe-Lys-NH₂. In one embodiment,the formulation is soluble in the cornea, aqueous humor, and lens of theeye. In one embodiment, the formulation further comprises apreservative. In one embodiment, the preservative is present in aconcentration of less than 1%.

In one embodiment, the formulation further comprises an active agentselected from the group consisting of: an antioxidant, a metalcomplexer, an anti-inflammatory drug, an antibiotic, and anantihistamine. In one embodiment, the antioxidant is vitamin A, vitaminC, vitamin E, lycopene, selenium, α-lipoic acid, coenzyme Q,glutathione, or a carotenoid. In one embodiment, the formulation furthercomprises an active agent selected from the group consisting of:aceclidine, acetazolamide, anecortave, apraclonidine, atropine,azapentacene, azelastine, bacitracin, befunolol, betamethasone,betaxolol, bimatoprost, brimonidine, brinzolamide, carbachol, carteolol,celecoxib, chloramphenicol, chlortetracycline, ciprofloxacin,cromoglycate, cromolyn, cyclopentolate, cyclosporin, dapiprazole,demecarium, dexamethasone, diclofenac, dichlorphenamide, dipivefrin,dorzolamide, echothiophate, emedastine, epinastine, epinephrine,erythromycin, ethoxzolamide, eucatropine, fludrocortisone,fluorometholone, flurbiprofen, fomivirsen, framycetin, ganciclovir,gatifloxacin, gentamycin, homatropine, hydrocortisone, idoxuridine,indomethacin, isoflurophate, ketorolac, ketotifen, latanoprost,levobetaxolol, levobunolol, levocabastine, levofloxacin, lodoxamide,loteprednol, medrysone, methazolamide, metipranolol, moxifloxacin,naphazoline, natamycin, nedocromil, neomycin, norfloxacin, ofloxacin,olopatadine, oxymetazoline, pemirolast, pegaptanib, phenylephrine,physostigmine, pilocarpine, pindolol, pirenoxine, polymyxin B,prednisolone, proparacaine, ranibizumab, rimexolone, scopolamine,sezolamide, squalamine, sulfacetamide, suprofen, tetracaine,tetracyclin, tetrahydrozoline, tetryzoline, timolol, tobramycin,travoprost, triamcinulone, trifluoromethazolamide, trifluridine,trimethoprim, tropicamide, unoprostone, vidarbine, xylometazoline,pharmaceutically acceptable salts thereof, and combinations thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the effects of different concentrations of SS-31(10 nM, 100 nM, 1 μM and 10 μM) used as co-treatment with 30 mM glucose(HG). FIG. 1A shows analysis for apoptosis, as assessed by a Flowcytometry after Annexin V/PI staining, which showed that the survivalratios for HRECs (Q3) was 99.3%, 83.2%, 84.3%, 90.7%, 92.8%, and 94.3%,respectively 24 hours after treatment. FIG. 1B is a graphicrepresentation of the survival ratio for HRECs. Data for SS-31concentrations of 100 nM, 1 μM, and 10 μM were significantly higher thanthat seen with high glucose exposed cells in the absence of co-treatmentwith SS31. * p<0.05 vs. 30 mM high glucose treated group.

FIGS. 2A-2F is a series of micrographs showing co-treatment with SS-31reduced intracellular reactive oxygen species (ROS) in HRECs exposed to30 mM glucose for 24 h and 48 h. Intracellular ROS was measured usingdihydroethidium. 2A, 2D normal culture media; 2B, 2E 30 mM glucose; and2C, 2F 30 mM glucose+SS-31 (100 nM) at 24 and 48 h, respectively.

FIGS. 3A and 3B show that SS-31 prevents the mitochondrial potentialloss of HRECs treated with high-glucose. FIG. 3A. The ΔΨm of HRECs wasmeasured by flow cytometry after JC-1 fluorescent probe staining. Highglucose (30 mM) treatment resulted in a rapid loss of mitochondrialmembrane potential of the cultured HRECs at 24 and 48 hours. Incontrast, flow cytometric analysis showed that 30 mM glucose co-treatedwith SS-31 increased ΔΨ_(m) compared with the high glucose alone group.FIG. 3B. Quantitative analysis of ΔΨm in high glucose HRECs co-treatedwith SS-31 for 24 and 48 hours, High glucose alone adversely affectedΔΨm. In contrast, SS-31 restored ΔΨm to control levels. Values representmean±SD of six separate experiments performed in triplicate. *P<0.05.

FIGS. 4A and 4D are confocal microscopic images showing that HRECs inthe normal glucose group and the SS-31 co-treated group have more exactoverlapping cytochrome c staining and HSP60 staining at 24 and 48 hours,indicating the co-localization of cytochrome c and mitochondria. Twentyfour and 48 hours after treatment, cytochrome c was obviously increasedin the cytoplasm of HRECs treated with 30 mM glucose. FIGS. 4B and 4Eshow the cytochrome c content in mitochondria and cytoplasm asdetermined by Western blot. FIGS. 4C and 4F show quantitative analysisof the percentage of cytochrome c content in mitochondria and cytoplasmof HRECs co-treated with high glucose and SS-31 for 24 and 48 h.

FIG. 5A and FIG. 5B show increased expression of caspase-3 in HRECstreated with high glucose (HG) was reduced by SS-31 co-treatment asdetected by western blot. Caspase-3 expression was normalized to theexpression of β-actin. FIGS. 5C-E show SS-31 increases the expression ofTrx2 in the high glucose-treated HRECs. FIG. 5C shows the mRNA level ofTrx2 in HRECs exposed to 30 mM glucose treated with SS-31 for 24 h and48 h. FIG. 5D shows the level of Trx2 protein expression measured byWestern blot. FIG. 5E shows quantitative analysis of the protein levelof Trx2 in HRECs 24 and 48 h after high glucose with or without SS-31co-treatment.

FIG. 6 is a photograph of the effects of SS-31 on the lenses of diabeticrats. Top row: lenses obtained from diabetic rats; bottom row: lensesobtained from diabetic rats treated with SS-31 or SS-20.

FIG. 7 is a series of photographs showing the effects of SS-31 and SS-20on the lenses of diabetic rats. Diabetes was induced by high fat dietand streptozotocin (HFD/STZ) (top row) or streptozotocin (STZ) alone(bottom row).

FIG. 8 is a series of micrographs showing the lens epithelium fromnormal rats, diabetic rats, and diabetic rats treated with SS-31.Diabetes was induced by STZ.

FIG. 9 is a series of micrographs showing the lens epithelium fromnormal rats, diabetic rats, and diabetic rats treated with SS-31.Diabetes was induced by HFD/STZ.

FIGS. 10A-10B is a series of charts showing the integrity of theblood-retinal barrier of normal rats (NRC), diabetic rats, and diabeticrats treated with SS-20 or SS-31, as analyzed by Evans blueextravasation. (A) diabetes induced by STZ; (B) diabetes induced byHFD/STZ.

FIG. 11 is a series of micrographs showing retinal microvessels ofnormal rats (NRC), diabetic rats (HFD/STZ), and diabetic rats treatedwith SS-31.

FIG. 12 is a series of micrographs showing retinal microvessels ofnormal rats, diabetic rats (STZ), and diabetic rats treated with SS-31.

FIGS. 13A-13D is a series of micrographs showing the distribution of thetight junction protein claudin-5 in retinal microvessels in normal rats(A), STZ rats (B), STZ/SS-20-treated rats (C), or STZ/SS-31-treated rats(D).

FIG. 14 is a chart showing the lack of cytotoxicity of SS-31 ontrabecular meshwork cells from non-diseased individuals (HTM) andtrabecular meshwork cells from glaucoma patients (GTM) administeredSS-31.

FIG. 15 is a series of confocal micrographs showing co-treatment withSS-31 dose-dependently inhibited the decrease in mitochondrial potential(Δψm) elicited by 200 μM H₂O₂ in trabecular meshwork cells from glaucomapatients (GTM).

FIGS. 16A and 16B is a series of charts showing co-treatment with SS-31inhibited the decrease in mitochondrial membrane potential (Δψm), asmeasured by TMRM and flow cytometry, in trabecular meshwork cells fromglaucoma patients (GTM) induced by 200 μM H₂O₂.

FIG. 17 is a chart comparing mitochondrial membrane potential (Δψm) inGTM and HTM cells.

FIG. 18 is a series of micrographs showing the morphology changes in GTMcells in response to SS-31 treatment as viewed using inverted phasecontrast microscopy.

FIG. 19 is a series of micrographs showing co-treatment with SS-31reduced the loss of mitochondrial membrane potential in GTM cells causedby 400 μM H₂O₂ in a dose-dependent manner as viewed using confocalmicroscopy.

FIG. 20 is a series of micrographs showing co-treatment with SS-31reduced the loss of mitochondrial membrane potential (Δψm) in GTM cellscaused by 400 μM H₂O₂ as viewed by TMRM and confocal microscopy (200×magnification).

FIG. 21 is a series of micrographs showing the morphology changes in GTMcells in response to SS-31 treatment as viewed using inverted phasecontrast microscopy.

FIG. 22 is a chart showing that SS-31 had no effect on the viability ofprimary human retinal pigment epithelial (RPE) cells (as measured by theMTT assay).

FIG. 23A is a chart showing the effect of different concentrations oftBHP on the viability (as measured by an MTT assay) of RPE cells. FIG.23B is a chart showing the effects of different concentrations of SS-31on cell viability when exposed to increasing concentrations of tBHP.

FIG. 24A-24C is a series of micrographs illustrating the pathologicaleffects in a choroidal neovascularization (CNV) mouse model. FIG. 24D isa graph showing CNV area in treated and control groups.

FIG. 25 is a series of micrographs illustrating different pathologicalfindings in an oxygen-induced retinopathy (OIR) mouse model. Note areasof avascularity and new vascularization in a P17 OIR mouse as comparedto a P17 normal mouse.

FIG. 26A-26D is a series of micrographs showing the effects ofadministering SS-31 in the OIR mouse model. FIG. 26E is a graph showingthe neovascular area of the control and treated groups. SS-31 reducedthe avascular area.

FIG. 27A is a chart showing the effect of different doses of tBHP oncell viability of a 661W cone cell line derived from a mouse retinaltumor. FIG. 27B is a chart showing the effect of 1 μM SS-31 in reducingtBHP-induced 661W cell death.

FIG. 28 is a series of micrographs showing the thickness of the retinalouter nuclear layer (ONL) in a mouse model of retina degeneration incontrol and SS-31-treated mice.

FIG. 29 is a series of micrographs showing the cone cell density inretinal flat mounts stained with peanut agglutinin (PNA), whichselectively stains cone inner and outer segments in control andSS-31-treated mice.

FIG. 30 is a series of micrographs showing staining for acolein, amarker for oxidative lipid damage in a mouse model of retinadegeneration.

FIGS. 31A-31D is a series of graphs showing fluorescence intensity ofintracellular ROS production in three groups of RPE cells using FACSanalysis. FIG. 31A shows ROS production in control RPE cells; FIG. 31Bshows ROS production in RPE cells treated with 500 μM tBHP for 3 h; FIG.31C shows ROS production in RPE cells treated with 500 μM tBHP for 3 hand 1 μM SS-31.

FIG. 32 is a series of graphs showing analysis of MMP labeled by JC-1 ina FACS assay. Three different concentration of SS-31 groups wereanalyzed.

FIGS. 33A-33D is a series of graphs showing the effect of 1 μM SS-31 onMMP decline induced by tBHP. FIG. 33A: Control group; FIG. 33B: 500 μMtBHP for 3 h group;

FIG. 33C: 1 μM SS-31 for 4 h+500 μM tBHP for 3 h group. FIG. 33D is achart comparing the fluorescence ratio for the different groups.*P<0.01, C vs. B.

FIGS. 34A-34D is a series of graphs showing the effect of SS-31 on cellapoptosis induced by 250 μM tBHP for 24 h. FIG. 34A: control group; FIG.34B: 250 μM tBHP for 24 h group; FIG. 34C: 1 μM SS-31 for 4 h+250 μMtBHP for 24 h group. FIG. 34D is a chart comparing the fluorescenceratio for the different groups. *P<0.05 C vs. B.

FIG. 35 is a chart showing the MDA level induced by tBHP in 3 groups ofRPE cells. *P<0.05, 1 μM SS-31 for 4 h+250 μM tBHP for 24 h group vs 250μM tBHP for 24 h.

FIG. 36 is a graph showing the fluorescence intensity of TMRM of GTM andHTM cells in control and SS-31-treated groups, as measured using FACSanalysis.

FIG. 37 is a graph showing the fluorescence intensity of ROS of GTM andHTM cells in control and SS-31-treated groups, as measured using FACSanalysis.

FIGS. 38A-38D is a series of graphs showing cell apoptosis of controland SS-31-treated groups valued by percentage of cells in the Q2+Q4quadrant.

FIGS. 39A-39B is a series of graphs showing that SS-31 reducedintracellular ROS production in GTM3 and iHTM cells treated with H₂O₂.

FIGS. 40A-40B is a series of graphs showing that SS-31 protected againstH₂O₂-induced mitochondrial depolarization of GTM3 and iHTM cells.

DETAILED DESCRIPTION

It is to be appreciated that certain aspects, modes, embodiments,variations and features of the invention are described below in variouslevels of detail in order to provide a substantial understanding of thepresent invention.

In practicing the present invention, many conventional techniques inmolecular biology, protein biochemistry, cell biology, immunology,microbiology and recombinant DNA are used. These techniques arewell-known and are explained in, e.g., Current Protocols in MolecularBiology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al., MolecularCloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989); DNA Cloning: A PracticalApproach, Vols. I and II, Glover, Ed. (1985); Oligonucleotide Synthesis,Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds.(1985); Transcription and Translation, Hames & Higgins, Eds. (1984);Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes(IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; theseries, Meth. Enzymol., (Academic Press, Inc., 1984); Gene TransferVectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring HarborLaboratory, N Y, 1987); and Meth. Enzymol., Vols. 154 and 155, Wu &Grossman, and Wu, Eds., respectively.

The definitions of certain terms as used in this specification areprovided below. Unless defined otherwise, all technical and scientificterms used herein generally have the same meaning as commonly understoodby one of ordinary skill in the art to which this invention belongs.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a cell” includesa combination of two or more cells, and the like.

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent depending upon the context inwhich it is used. If there are uses of the term which are not clear topersons of ordinary skill in the art, given the context in which it isused, “about” will mean up to plus or minus 10% of the enumerated value.

As used herein, the “administration” of an agent, drug, or peptide to asubject includes any route of introducing or delivering to a subject acompound to perform its intended function. Administration can be carriedout by any suitable route, including orally, intraocularly,intranasally, parenterally (intravenously, intramuscularly,intraperitoneally, or subcutaneously), or topically. Administrationincludes self-administration and the administration by another.

As used herein, the term “amino acid” includes naturally-occurring aminoacids and synthetic amino acids, as well as amino acid analogs and aminoacid mimetics that function in a manner similar to thenaturally-occurring amino acids. Naturally-occurring amino acids arethose encoded by the genetic code, as well as those amino acids that arelater modified, e.g., hydroxyproline, γ-carboxyglutamate, andO-phosphoserine. Amino acid analogs refers to compounds that have thesame basic chemical structure as a naturally-occurring amino acid, i.e.,an α-carbon that is bound to a hydrogen, a carboxyl group, an aminogroup, and an R group, e.g., homoserine, norleucine, methioninesulfoxide, methionine methyl sulfonium. Such analogs have modified Rgroups (e.g., norleucine) or modified peptide backbones, but retain thesame basic chemical structure as a naturally-occurring amino acid. Aminoacid mimetics refers to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunctions in a manner similar to a naturally-occurring amino acid. Aminoacids can be referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission.

As used herein, the term “effective amount” refers to a quantitysufficient to achieve a desired therapeutic and/or prophylactic effect,e.g., an amount which results in the prevention of, or a decrease in,the symptoms associated with an ophthalmic condition. The amount of acomposition administered to the subject will depend on the type andseverity of the disease and on the characteristics of the individual,such as general health, age, sex, body weight and tolerance to drugs. Itwill also depend on the degree, severity and type of disease. Theskilled artisan will be able to determine appropriate dosages dependingon these and other factors. The compositions can also be administered incombination with one or more additional therapeutic compounds. In themethods described herein, the aromatic-cationic peptides may beadministered to a subject having one or more signs or symptoms of anophthalmic condition. For example, a “therapeutically effective amount”of the aromatic-cationic peptides is meant levels in which thephysiological effects of an ophthalmic condition are, at a minimum,ameliorated.

An “isolated” or “purified” polypeptide or peptide is substantially freeof cellular material or other contaminating polypeptides from the cellor tissue source from which the agent is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.For example, an isolated aromatic-cationic peptide would be free ofmaterials that would interfere with diagnostic or therapeutic uses ofthe agent. Such interfering materials may include enzymes, hormones andother proteinaceous and nonproteinaceous solutes.

As used herein, the terms “polypeptide”, “peptide” and “protein” areused interchangeably herein to mean a polymer comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. Polypeptide refers to both short chains,commonly referred to as peptides, glycopeptides or oligomers, and tolonger chains, generally referred to as proteins. Polypeptides maycontain amino acids other than the 20 gene-encoded amino acids.Polypeptides include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques that are well known in the art.

As used herein, the term “simultaneous” therapeutic use refers to theadministration of at least two active ingredients by the same route andat the same time or at substantially the same time.

As used herein, the term “separate” therapeutic use refers to anadministration of at least two active ingredients at the same time or atsubstantially the same time by different routes.

As used herein, the term “sequential” therapeutic use refers toadministration of at least two active ingredients at different times,the administration route being identical or different. Moreparticularly, sequential use refers to the whole administration of oneof the active ingredients before administration of the other or otherscommences. It is thus possible to administer one of the activeingredients over several minutes, hours, or days before administeringthe other active ingredient or ingredients. There is no simultaneoustreatment in this case.

As used herein, the terms “treating” or “treatment” or “alleviation”refers to both therapeutic treatment and prophylactic or preventativemeasures, wherein the object is to prevent or slow down (lessen) thetargeted pathologic condition or disorder. A subject is successfully“treated” for an ophthalmic condition if, after receiving a therapeuticamount of the aromatic-cationic peptides according to the methodsdescribed herein, the subject shows observable and/or measurablereduction in or absence of one or more signs and symptoms of anophthalmic condition. It is also to be appreciated that the variousmodes of treatment or prevention of medical conditions as described areintended to mean “substantial”, which includes total but also less thantotal treatment or prevention, and wherein some biologically ormedically relevant result is achieved.

As used herein, “prevention” or “preventing” of a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample.

Aromatic-Cationic Peptides

The present technology relates to the treatment or prevention of anophthalmic condition by administration of certain aromatic-cationicpeptides. Without wishing to be limited by theory, the aromatic-cationicpeptides may treat or prevent ophthalmic diseases or conditions byreducing the severity or occurrence of oxidative damage in the eye. Thearomatic-cationic peptides are water-soluble and highly polar. Despitethese properties, the peptides can readily penetrate cell membranes. Thearomatic-cationic peptides typically include a minimum of three aminoacids or a minimum of four amino acids, covalently joined by peptidebonds. The maximum number of amino acids present in thearomatic-cationic peptides is about twenty amino acids covalently joinedby peptide bonds. Suitably, the maximum number of amino acids is abouttwelve, more preferably about nine, and most preferably about six.

The amino acids of the aromatic-cationic peptides can be any amino acid.As used herein, the term “amino acid” is used to refer to any organicmolecule that contains at least one amino group and at least onecarboxyl group. Typically, at least one amino group is at the α positionrelative to a carboxyl group. The amino acids may be naturallyoccurring. Naturally occurring amino acids include, for example, thetwenty most common levorotatory (L) amino acids normally found inmammalian proteins, i.e., alanine (Ala), arginine (Arg), asparagine(Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamicacid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine(Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline(Pro), serine (Ser), threonine (Thr), tryptophan, (Trp), tyrosine (Tyr),and valine (Val). Other naturally occurring amino acids include, forexample, amino acids that are synthesized in metabolic processes notassociated with protein synthesis. For example, the amino acidsornithine and citrulline are synthesized in mammalian metabolism duringthe production of urea. Another example of a naturally occurring aminoacid include hydroxyproline (Hyp).

The peptides optionally contain one or more non-naturally occurringamino acids. Suitably, the peptide has no amino acids that are naturallyoccurring. The non-naturally occurring amino acids may be levorotary(L-), dextrorotatory (D-), or mixtures thereof. Non-naturally occurringamino acids are those amino acids that typically are not synthesized innormal metabolic processes in living organisms, and do not naturallyoccur in proteins. In addition, the non-naturally occurring amino acidssuitably are also not recognized by common proteases. The non-naturallyoccurring amino acid can be present at any position in the peptide. Forexample, the non-naturally occurring amino acid can be at theN-terminus, the C-terminus, or at any position between the N-terminusand the C-terminus.

The non-natural amino acids may, for example, comprise alkyl, aryl, oralkylaryl groups not found in natural amino acids. Some examples ofnon-natural alkyl amino acids include α-aminobutyric acid,β-aminobutyric acid, γ-aminobutyric acid, δ-aminovaleric acid, andε-aminocaproic acid. Some examples of non-natural aryl amino acidsinclude ortho-, meta, and para-aminobenzoic acid. Some examples ofnon-natural alkylaryl amino acids include ortho-, meta-, andpara-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid.Non-naturally occurring amino acids include derivatives of naturallyoccurring amino acids. The derivatives of naturally occurring aminoacids may, for example, include the addition of one or more chemicalgroups to the naturally occurring amino acid.

For example, one or more chemical groups can be added to one or more ofthe 2′, 3′, 4′, 5′, or 6′ position of the aromatic ring of aphenylalanine or tyrosine residue, or the 4′, 5′, 6′, or 7′ position ofthe benzo ring of a tryptophan residue. The group can be any chemicalgroup that can be added to an aromatic ring. Some examples of suchgroups include branched or unbranched C₁-C₄ alkyl, such as methyl,ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C₁-C₄ alkyloxy(i.e., alkoxy), amino, C₁-C₄ alkylamino and C₁-C₄ dialkylamino (e.g.,methylamino, dimethylamino), nitro, hydroxyl, halo (i.e., fluoro,chloro, bromo, or iodo). Some specific examples of non-naturallyoccurring derivatives of naturally occurring amino acids includenorvaline (Nva) and norleucine (Nle).

Another example of a modification of an amino acid in a peptide is thederivatization of a carboxyl group of an aspartic acid or a glutamicacid residue of the peptide. One example of derivatization is amidationwith ammonia or with a primary or secondary amine, e.g. methylamine,ethylamine, dimethylamine or diethylamine. Another example ofderivatization includes esterification with, for example, methyl orethyl alcohol. Another such modification includes derivatization of anamino group of a lysine, arginine, or histidine residue. For example,such amino groups can be acylated. Some suitable acyl groups include,for example, a benzoyl group or an alkanoyl group comprising any of theC₁-C₄ alkyl groups mentioned above, such as an acetyl or propionylgroup.

The non-naturally occurring amino acids are preferably resistant, andmore preferably insensitive, to common proteases. Examples ofnon-naturally occurring amino acids that are resistant or insensitive toproteases include the dextrorotatory (D-) form of any of theabove-mentioned naturally occurring L-amino acids, as well as L- and/orD-non-naturally occurring amino acids. The D-amino acids do not normallyoccur in proteins, although they are found in certain peptideantibiotics that are synthesized by means other than the normalribosomal protein synthetic machinery of the cell. As used herein, theD-amino acids are considered to be non-naturally occurring amino acids.

In order to minimize protease sensitivity, the peptides should have lessthan five, preferably less than four, more preferably less than three,and most preferably, less than two contiguous L-amino acids recognizedby common proteases, irrespective of whether the amino acids arenaturally or non-naturally occurring. Suitably, the peptide has onlyD-amino acids, and no L-amino acids. If the peptide contains proteasesensitive sequences of amino acids, at least one of the amino acids ispreferably a non-naturally-occurring D-amino acid, thereby conferringprotease resistance. An example of a protease sensitive sequenceincludes two or more contiguous basic amino acids that are readilycleaved by common proteases, such as endopeptidases and trypsin.Examples of basic amino acids include arginine, lysine and histidine.

The aromatic-cationic peptides should have a minimum number of netpositive charges at physiological pH in comparison to the total numberof amino acid residues in the peptide. The minimum number of netpositive charges at physiological pH will be referred to below as(p_(m)). The total number of amino acid residues in the peptide will bereferred to below as (r). The minimum number of net positive chargesdiscussed below are all at physiological pH. The term “physiological pH”as used herein refers to the normal pH in the cells of the tissues andorgans of the mammalian body. For instance, the physiological pH of ahuman is normally approximately 7.4, but normal physiological pH inmammals may be any pH from about 7.0 to about 7.8.

“Net charge” as used herein refers to the balance of the number ofpositive charges and the number of negative charges carried by the aminoacids present in the peptide. In this specification, it is understoodthat net charges are measured at physiological pH. The naturallyoccurring amino acids that are positively charged at physiological pHinclude L-lysine, L-arginine, and L-histidine. The naturally occurringamino acids that are negatively charged at physiological pH includeL-aspartic acid and L-glutamic acid.

Typically, a peptide has a positively charged N-terminal amino group anda negatively charged C-terminal carboxyl group. The charges cancel eachother out at physiological pH. As an example of calculating net charge,the peptide Tyr-Arg-Phe-Lys-Glu-His-Trp-D-Arg has one negatively chargedamino acid (i.e., Glu) and four positively charged amino acids (i.e.,two Arg residues, one Lys, and one His). Therefore, the above peptidehas a net positive charge of three.

In one embodiment, the aromatic-cationic peptides have a relationshipbetween the minimum number of net positive charges at physiological pH(p_(m)) and the total number of amino acid residues (r) wherein 3p_(m)is the largest number that is less than or equal to r+1. In thisembodiment, the relationship between the minimum number of net positivecharges (p_(m)) and the total number of amino acid residues (r) is asfollows:

TABLE 1 Amino acid number and net positive charges (3p_(m) ≤ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 1 1 2 2 2 3 3 3 44 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) wherein 2p_(m) is thelargest number that is less than or equal to r+1. In this embodiment,the relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) is as follows:

TABLE 2 Amino acid number and net positive charges (2p_(m) ≤ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 2 2 3 3 4 4 5 5 66 7 7 8 8 9 9 10 10

In one embodiment, the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) are equal. In anotherembodiment, the peptides have three or four amino acid residues and aminimum of one net positive charge, preferably, a minimum of two netpositive charges and more preferably a minimum of three net positivecharges.

It is also important that the aromatic-cationic peptides have a minimumnumber of aromatic groups in comparison to the total number of netpositive charges (p_(t)). The minimum number of aromatic groups will bereferred to below as (a). Naturally occurring amino acids that have anaromatic group include the amino acids histidine, tryptophan, tyrosine,and phenylalanine. For example, the hexapeptideLys-Gln-Tyr-D-Arg-Phe-Trp has a net positive charge of two (contributedby the lysine and arginine residues) and three aromatic groups(contributed by tyrosine, phenylalanine and tryptophan residues).

The aromatic-cationic peptides should also have a relationship betweenthe minimum number of aromatic groups (a) and the total number of netpositive charges at physiological pH (p_(t)) wherein 3a is the largestnumber that is less than or equal to p_(t)+1, except that when p_(t) is1, a may also be 1. In this embodiment, the relationship between theminimum number of aromatic groups (a) and the total number of netpositive charges (p_(t)) is as follows:

TABLE 3 Aromatic groups and net positive charges (3a ≤ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1. In this embodiment, therelationship between the minimum number of aromatic amino acid residues(a) and the total number of net positive charges (p_(t)) is as follows:

TABLE 4 Aromatic groups and net positive charges (2a ≤ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10

In another embodiment, the number of aromatic groups (a) and the totalnumber of net positive charges (p_(t)) are equal.

Carboxyl groups, especially the terminal carboxyl group of a C-terminalamino acid, are preferably amidated with, for example, ammonia to formthe C-terminal amide. Alternatively, the terminal carboxyl group of theC-terminal amino acid may be amidated with any primary or secondaryamine. The primary or secondary amine may, for example, be an alkyl,especially a branched or unbranched C₁-C₄ alkyl, or an aryl amine.Accordingly, the amino acid at the C-terminus of the peptide may beconverted to an amido, N-methylamido, N-ethylamido, N,N-dimethylamido,N,N-diethylamido, N-methyl-N-ethylamido, N-phenylamido orN-phenyl-N-ethylamido group. The free carboxylate groups of theasparagine, glutamine, aspartic acid, and glutamic acid residues notoccurring at the C-terminus of the aromatic-cationic peptides may alsobe amidated wherever they occur within the peptide. The amidation atthese internal positions may be with ammonia or any of the primary orsecondary amines described above.

In one embodiment, the aromatic-cationic peptide is a tripeptide havingtwo net positive charges and at least one aromatic amino acid. In aparticular embodiment, the aromatic-cationic peptide is a tripeptidehaving two net positive charges and two aromatic amino acids.

Aromatic-cationic peptides include, but are not limited to, thefollowing peptide examples:

-   -   Lys-D-Arg-Tyr-NH₂    -   Phe-D-Arg-His    -   D-Tyr-Trp-Lys-NH₂    -   Trp-D-Lys-Tyr-Arg-NH₂    -   Tyr-His-D-Gly-Met    -   Phe-Arg-D-His-Asp    -   Tyr-D-Arg-Phe-Lys-Glu-NH₂    -   Met-Tyr-D-Lys-Phe-Arg    -   D-His-Glu-Lys-Tyr-D-Phe-Arg    -   Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH₂    -   Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His    -   Gly-D-Phe-Lys-Tyr-His-D-Arg-Tyr-NH₂    -   Val-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH₂    -   Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-Lys    -   Lys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH₂    -   Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-Lys    -   Asp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH₂    -   D-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-His-D-Lys-Arg-Trp-NH₂    -   Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-Phe    -   Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-Phe    -   Phe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH₂    -   Phe-Try-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-Thr    -   Tyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr-His-Lys    -   Glu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg-D-Met-NH₂    -   Arg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-Phe-Tyr-D-Arg-Gly    -   D-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH₂    -   Asp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-Phe    -   His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH₂    -   Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-Asp    -   Thr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg-Tyr-Lys-NH₂

In one embodiment, a peptide that has mu-opioid receptor agonistactivity has the formula Tyr-D-Arg-Phe-Lys-NH₂ (referred to herein as“SS-01”). SS-01 has a net positive charge of three, contributed by theamino acids tyrosine, arginine, and lysine and has two aromatic groupscontributed by the amino acids phenylalanine and tyrosine. The tyrosineof SS-01 can be a modified derivative of tyrosine such as in2′,6′-dimethyltyrosine to produce the compound having the formula2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ (referred to herein as “SS-02”). SS-02 has amolecular weight of 640 and carries a net three positive charge atphysiological pH. SS-02 readily penetrates the plasma membrane ofseveral mammalian cell types in an energy-independent manner (Zhao etal., J. Pharmacol Exp Ther. 304: 425-432, 2003).

Peptides that do not have mu-opioid receptor agonist activity generallydo not have a tyrosine residue or a derivative of tyrosine at theN-terminus (i.e., amino acid position 1). The amino acid at theN-terminus can be any naturally occurring or non-naturally occurringamino acid other than tyrosine. In one embodiment, the amino acid at theN-terminus is phenylalanine or its derivative. Exemplary derivatives ofphenylalanine include 2′-methylphenylalanine (Mmp),2′,6′-dimethylphenylalanine (2′,6′-Dmp), N,2′,6′-trimethylphenylalanine(Tmp), and 2′-hydroxy-6′-methylphenylalanine (Hmp).

An example of an aromatic-cationic peptide that does not have mu-opioidreceptor agonist activity has the formula Phe-D-Arg-Phe-Lys-NH₂(referred to herein as “SS-20”). Alternatively, the N-terminalphenylalanine can be a derivative of phenylalanine such as2′,6′-dimethylphenylalanine (2′6′-Dmp). SS-01 containing2′,6′-dimethylphenylalanine at amino acid position 1 has the formula2′,6′-Dmp-D-Arg-Phe-Lys-NH₂. In one embodiment, the amino acid sequenceof SS-02 is rearranged such that Dmt is not at the N-terminus. Anexample of such an aromatic-cationic peptide that does not havemu-opioid receptor agonist activity has the formulaD-Arg-2′6′-Dmt-Lys-Phe-NH₂ (SS-31).

SS-01, SS-20, SS-31, and their derivatives can further includefunctional analogs. A peptide is considered a functional analog ofSS-01, SS-20, or SS-31 if the analog has the same function as SS-01,SS-20, or SS-31. The analog may, for example, be a substitution variantof SS-01, SS-20, or SS-31, wherein one or more amino acids aresubstituted by another amino acid.

Suitable substitution variants of SS-01, SS-20, or SS-31 includeconservative amino acid substitutions. Amino acids may be groupedaccording to their physicochemical characteristics as follows:

(a) Non-polar amino acids: Ala(A) Ser(S) Thr(T) Pro(P) Gly(G) Cys (C);

(b) Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(Q);

(c) Basic amino acids: His(H) Arg(R) Lys(K);

(d) Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V); and

(e) Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His (H).

Substitutions of an amino acid in a peptide by another amino acid in thesame group is referred to as a conservative substitution and maypreserve the physicochemical characteristics of the original peptide. Incontrast, substitutions of an amino acid in a peptide by another aminoacid in a different group is generally more likely to alter thecharacteristics of the original peptide.

In some embodiments, the aromatic-cationic peptide has a formula asshown in Table 5.

TABLE 5 Peptide Analogs with Mu-Opioid Activity Amino Acid Amino AcidAmino Acid Amino Acid C-Terminal Position 1 Position 2 Position 3Position 4 Modification Tyr D-Arg Phe Lys NH₂ Tyr D-Arg Phe Orn NH₂ TyrD-Arg Phe Dab NH₂ Tyr D-Arg Phe Dap NH₂ 2′6′Dmt D-Arg Phe Lys NH₂2′6′Dmt D-Arg Phe Lys-NH(CH₂)₂—NH-dns NH₂ 2′6′Dmt D-Arg PheLys-NH(CH₂)₂—NH-atn NH₂ 2′6′Dmt D-Arg Phe dnsLys NH₂ 2′6′Dmt D-Cit PheLys NH₂ 2′6′Dmt D-Cit Phe Ahp NH₂ 2′6′Dmt D-Arg Phe Orn NH₂ 2′6′DmtD-Arg Phe Dab NH₂ 2′6′Dmt D-Arg Phe Dap NH₂ 2′6′Dmt D-Arg PheAhp(2-aminoheptanoic acid) NH₂ Bio-2′6′Dmt D-Arg Phe Lys NH₂ 3′5′DmtD-Arg Phe Lys NH₂ 3′5′Dmt D-Arg Phe Orn NH₂ 3′5′Dmt D-Arg Phe Dab NH₂3′5′Dmt D-Arg Phe Dap NH₂ Tyr D-Arg Tyr Lys NH₂ Tyr D-Arg Tyr Orn NH₂Tyr D-Arg Tyr Dab NH₂ Tyr D-Arg Tyr Dap NH₂ 2′6′Dmt D-Arg Tyr Lys NH₂2′6′Dmt D-Arg Tyr Orn NH₂ 2′6′Dmt D-Arg Tyr Dab NH₂ 2′6′Dmt D-Arg TyrDap NH₂ 2′6′Dmt D-Arg 2′6′Dmt Lys NH₂ 2′6′Dmt D-Arg 2′6′Dmt Orn NH₂2′6′Dmt D-Arg 2′6′Dmt Dab NH₂ 2′6′Dmt D-Arg 2′6′Dmt Dap NH₂ 3′5′DmtD-Arg 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg 3′5′Dmt Lys NH₂ 3′5′Dmt D-Arg3′5′Dmt Orn NH₂ 3′5′Dmt D-Arg 3′5′Dmt Dab NH₂ Tyr D-Lys Phe Dap NH₂ TyrD-Lys Phe Arg NH₂ Tyr D-Lys Phe Lys NH₂ Tyr D-Lys Phe Orn NH₂ 2′6′DmtD-Lys Phe Dab NH₂ 2′6′Dmt D-Lys Phe Dap NH₂ 2′6′Dmt D-Lys Phe Arg NH₂2′6′Dmt D-Lys Phe Lys NH₂ 3′5′Dmt D-Lys Phe Orn NH₂ 3′5′Dmt D-Lys PheDab NH₂ 3′5′Dmt D-Lys Phe Dap NH₂ 3′5′Dmt D-Lys Phe Arg NH₂ Tyr D-LysTyr Lys NH₂ Tyr D-Lys Tyr Orn NH₂ Tyr D-Lys Tyr Dab NH₂ Tyr D-Lys TyrDap NH₂ 2′6′Dmt D-Lys Tyr Lys NH₂ 2′6′Dmt D-Lys Tyr Orn NH₂ 2′6′DmtD-Lys Tyr Dab NH₂ 2′6′Dmt D-Lys Tyr Dap NH₂ 2′6′Dmt D-Lys 2′6′Dmt LysNH₂ 2′6′Dmt D-Lys 2′6′Dmt Orn NH₂ 2′6′Dmt D-Lys 2′6′Dmt Dab NH₂ 2′6′DmtD-Lys 2′6′Dmt Dap NH₂ 2′6′Dmt D-Arg Phe dnsDap NH₂ 2′6′Dmt D-Arg PheatnDap NH₂ 3′5′Dmt D-Lys 3′5′Dmt Lys NH₂ 3′5′Dmt D-Lys 3′5′Dmt Orn NH₂3′5′Dmt D-Lys 3′5′Dmt Dab NH₂ 3′5′Dmt D-Lys 3′5′Dmt Dap NH₂ Tyr D-LysPhe Arg NH₂ Tyr D-Orn Phe Arg NH₂ Tyr D-Dab Phe Arg NH₂ Tyr D-Dap PheArg NH₂ 2′6′Dmt D-Arg Phe Arg NH₂ 2′6′Dmt D-Lys Phe Arg NH₂ 2′6′DmtD-Orn Phe Arg NH₂ 2′6′Dmt D-Dab Phe Arg NH₂ 3′5′Dmt D-Dap Phe Arg NH₂3′5′Dmt D-Arg Phe Arg NH₂ 3′5′Dmt D-Lys Phe Arg NH₂ 3′5′Dmt D-Orn PheArg NH₂ Tyr D-Lys Tyr Arg NH₂ Tyr D-Orn Tyr Arg NH₂ Tyr D-Dab Tyr ArgNH₂ Tyr D-Dap Tyr Arg NH₂ 2′6′Dmt D-Arg 2′6′Dmt Arg NH₂ 2′6′Dmt D-Lys2′6′Dmt Arg NH₂ 2′6′Dmt D-Orn 2′6′Dmt Arg NH₂ 2′6′Dmt D-Dab 2′6′Dmt ArgNH₂ 3′5′Dmt D-Dap 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg 3′5′Dmt Arg NH₂ 3′5′DmtD-Lys 3′5′Dmt Arg NH₂ 3′5′Dmt D-Orn 3′5′Dmt Arg NH₂ Mmt D-Arg Phe LysNH₂ Mmt D-Arg Phe Orn NH₂ Mmt D-Arg Phe Dab NH₂ Mmt D-Arg Phe Dap NH₂Tmt D-Arg Phe Lys NH₂ Tmt D-Arg Phe Orn NH₂ Tmt D-Arg Phe Dab NH₂ TmtD-Arg Phe Dap NH₂ Hmt D-Arg Phe Lys NH₂ Hmt D-Arg Phe Orn NH₂ Hmt D-ArgPhe Dab NH₂ Hmt D-Arg Phe Dap NH₂ Mmt D-Lys Phe Lys NH₂ Mmt D-Lys PheOrn NH₂ Mmt D-Lys Phe Dab NH₂ Mmt D-Lys Phe Dap NH₂ Mmt D-Lys Phe ArgNH₂ Tmt D-Lys Phe Lys NH₂ Tmt D-Lys Phe Orn NH₂ Tmt D-Lys Phe Dab NH₂Tmt D-Lys Phe Dap NH₂ Tmt D-Lys Phe Arg NH₂ Hmt D-Lys Phe Lys NH₂ HmtD-Lys Phe Orn NH₂ Hmt D-Lys Phe Dab NH₂ Hmt D-Lys Phe Dap NH₂ Hmt D-LysPhe Arg NH₂ Mmt D-Lys Phe Arg NH₂ Mmt D-Orn Phe Arg NH₂ Mmt D-Dab PheArg NH₂ Mmt D-Dap Phe Arg NH₂ Mmt D-Arg Phe Arg NH₂ Tmt D-Lys Phe ArgNH₂ Tmt D-Orn Phe Arg NH₂ Tmt D-Dab Phe Arg NH₂ Tmt D-Dap Phe Arg NH₂Tmt D-Arg Phe Arg NH₂ Hmt D-Lys Phe Arg NH₂ Hmt D-Orn Phe Arg NH₂ HmtD-Dab Phe Arg NH₂ Hmt D-Dap Phe Arg NH₂ Hmt D-Arg Phe Arg NH₂ Dab =diaminobutyric Dap = diaminopropionic acid Dmt = dimethyltyrosine Mmt =2′-methyltyrosine Tmt = N,2′,6′-trimethyltyrosine Hmt =2′-hydroxy,6′-methyltyrosine dnsDap = β-dansyl-L-α,β-diaminopropionicacid atnDap = β-anthraniloyl-L-α,β-diaminopropionic acid Bio = biotin

Examples of other aromatic-cationic peptides that do not activatemu-opioid receptors include, but are not limited to, thearomatic-cationic peptides shown in Table 6.

TABLE 6 Peptide Analogs Lacking Mu-Opioid Activity Amino Acid Amino AcidAmino Acid Amino Acid C-Terminal Position 1 Position 2 Position 3Position 4 Modification D-Arg Dmt Lys Phe NH₂ D-Arg Dmt Phe Lys NH₂D-Arg Phe Lys Dmt NH₂ D-Arg Phe Dmt Lys NH₂ D-Arg Lys Dmt Phe NH₂ D-ArgLys Phe Dmt NH₂ Phe Lys Dmt D-Arg NH₂ Phe Lys D-Arg Dmt NH₂ Phe D-ArgPhe Lys NH₂ Phe D-Arg Dmt Lys NH₂ Phe D-Arg Lys Dmt NH₂ Phe Dmt D-ArgLys NH₂ Phe Dmt Lys D-Arg NH₂ Lys Phe D-Arg Dmt NH₂ Lys Phe Dmt D-ArgNH₂ Lys Dmt D-Arg Phe NH₂ Lys Dmt Phe D-Arg NH₂ Lys D-Arg Phe Dmt NH₂Lys D-Arg Dmt Phe NH₂ D-Arg Dmt D-Arg Phe NH₂ D-Arg Dmt D-Arg Dmt NH₂D-Arg Dmt D-Arg Tyr NH₂ D-Arg Dmt D-Arg Trp NH₂ Trp D-Arg Phe Lys NH₂Trp D-Arg Tyr Lys NH₂ Trp D-Arg Trp Lys NH₂ Trp D-Arg Dmt Lys NH₂ D-ArgTrp Lys Phe NH₂ D-Arg Trp Phe Lys NH₂ D-Arg Trp Lys Dmt NH₂ D-Arg TrpDmt Lys NH₂ D-Arg Lys Trp Phe NH₂ D-Arg Lys Trp Dmt NH₂ Cha D-Arg PheLys NH₂ Ala D-Arg Phe Lys NH₂ Cha = cyclohexyl alanine

The amino acids of the peptides shown in Table 5 and 6 may be in eitherthe L- or the D-configuration.

The peptides may be synthesized by any of the methods well known in theart. Suitable methods for chemically synthesizing the protein include,for example, those described by Stuart and Young in Solid Phase PeptideSynthesis, Second Edition, Pierce Chemical Company (1984), and inMethods Enzymol. 289, Academic Press, Inc, New York (1997).

Prophylactic and Therapeutic Uses of Aromatic-Cationic Peptides.

The aromatic-cationic peptides described herein are useful to prevent ortreat disease. Specifically, the disclosure provides for bothprophylactic and therapeutic methods of treating a subject at risk of(or susceptible to) an ophthalmic disease or condition. Accordingly, thepresent methods provide for the prevention and/or treatment of anophthalmic condition in a subject by administering an effective amountof an aromatic-cationic peptide to a subject in need thereof. Forexample, a subject can be administered an aromatic-cationic peptidecompositions in an effort to improve one or more of the factorscontributing to an ophthalmic disease or condition.

One aspect of the technology includes methods of reducing an ophthalmiccondition in a subject for therapeutic purposes. In therapeuticapplications, compositions or medicaments are administered to a subjectsuspected of, or already suffering from such a disease in an amountsufficient to cure, or at least partially arrest, the symptoms of thedisease, including its complications and intermediate pathologicalphenotypes in development of the disease. As such, the disclosureprovides methods of treating an individual afflicted with an ophthalmiccondition. In some embodiments, the technology provides a method oftreating or preventing specific ophthalmic disorders, such as diabeticretinopathy, cataracts, retinitis pigmentosa, glaucoma, choroidalneovascularization, retinal degeneration, and oxygen-inducedretinopathy, in a mammal by administering an aromatic cationic peptide.

In one embodiment, an aromatic-cationic peptide is administered to asubject to treat or prevent diabetic retinopathy. Diabetic retinopathyis characterized by capillary microaneurysms and dot hemorrhaging.Thereafter, microvascular obstructions cause cotton wool patches to formon the retina. Moreover, retinal edema and/or hard exudates may form inindividuals with diabetic retinopathy due to increased vascularhyperpermeability. Subsequently, neovascularization appears and retinaldetachment is caused by traction of the connective tissue grown in thevitreous body. Iris rubeosis and neovascular glaucoma may also occurwhich, in turn, can lead to blindness. The symptoms of diabeticretinopathy include, but are not limited to, difficulty reading, blurredvision, sudden loss of vision in one eye, seeing rings around lights,seeing dark spots, and/or seeing flashing lights.

In one embodiment, an aromatic-cationic peptide is administered to asubject to treat or prevent cataracts. Cataracts is a congenital oracquired disease characterized by a reduction in natural lens clarity.Individuals with cataracts may exhibit one or more symptoms, including,but not limited to, cloudiness on the surface of the lens, cloudiness onthe inside of the lens, and/or swelling of the lens. Typical examples ofcongenital cataract-associated diseases are pseudo-cataracts, membranecataracts, coronary cataracts, lamellar cataracts, punctuate cataracts,and filamentary cataracts. Typical examples of acquiredcataract-associated diseases are geriatric cataracts, secondarycataracts, browning cataracts, complicated cataracts, diabeticcataracts, and traumatic cataracts. Acquired cataracts is also inducibleby electric shock, radiation, ultrasound, drugs, systemic diseases, andnutritional disorders. Acquired cataracts further includes postoperativecataracts.

In one embodiment, an aromatic-cationic peptide is administered to asubject to treat or prevent retinitis pigmentosa. Retinitis pigmentosais a disorder that is characterized by rod and/or cone cell damage. Thepresence of dark lines in the retina is typical in individuals sufferingfrom retinitis pigmentosa. Individuals with retinitis pigmentosa alsopresent with a variety of symptoms including, but not limited to,headaches, numbness or tingling in the extremities, light flashes,and/or visual changes. See, e.g., Heckenlively et al., Clinical findingsand common symptoms in retinitis pigmentosa. Am J Ophthalmol. 105(5):504-511 (1988).

In one embodiment, an aromatic-cationic peptide is administered to asubject to treat or prevent glaucoma. Glaucoma is a genetic diseasecharacterized by an increase in intraocular pressure, which leads to adecrease in vision. Glaucoma may emanate from various ophthalmologicconditions that are already present in an individual, such as, wounds,surgery, and other structural malformations. Although glaucoma can occurat any age, it frequently develops in elderly individuals and leads toblindness. Glaucoma patients typically have an intraocular pressure inexcess of 21 mmHg. However, normal tension glaucoma, where glaucomatousalterations are found in the visual field and optic papilla, can occurin the absence of such increased intraocular pressures, i.e., greaterthan 21 mmHg. Symptoms of glaucoma include, but are not limited to,blurred vision, severe eye pain, headache, seeing haloes around lights,nausea, and/or vomiting.

In one embodiment, an aromatic-cationic peptide is administered to asubject to treat or prevent macular degeneration. Macular degenerationis typically an age-related disease. The general categories of maculardegeneration include wet, dry, and non-aged related maculardegeneration. Dry macular degeneration, which accounts for about 80-90percent of all cases, is also known as atrophic, nonexudative, ordrusenoid macular degeneration. With dry macular degeneration, drusentypically accumulate beneath the retinal pigment epithelium tissue.Vision loss subsequently occurs when drusen interfere with the functionof photoreceptors in the macula. Symptoms of dry macular generationinclude, but are not limited to, distorted vision, center-visiondistortion, light or dark distortion, and/or changes in colorperception. Dry macular degeneration can result in the gradual loss ofvision.

Wet macular degeneration is also known as neovascularization, subretinalneovascularization, exudative, or disciform degeneration. With wetmacular degeneration, abnormal blood vessels grow beneath the macula.The blood vessels leak fluid into the macula and damage photoreceptorcells. Wet macular degeneration can progress rapidly and cause severedamage to central vision. Wet and dry macular degeneration haveidentical symptoms. Non-age related macular degeneration, however, israre and may be linked to heredity, diabetes, nutritional deficits,injury, infection, or other factors. The symptoms of non-age relatedmacular degeneration also include, but are not limited to, distortedvision, center-vision distortion, light or dark distortion, and/orchanges in color perception.

In one embodiment, an aromatic-cationic peptide is administered to asubject to treat or prevent choroidal neovascularization. Choroidalneovascularization (CNV) is a disease characterized by the developmentof new blood vessels in the choroid layer of the eye. The newly formedblood vessels grow in the choroid, through the Bruch membrane, andinvade the subretinal space. CNV can lead to the impairment of sight orcomplete loss of vision. Symptoms of CNV include, but are not limitedto, seeing flickering, blinking lights, or gray spots in the affectedeye or eyes, blurred vision, distorted vision, and/or loss of vision.

In one embodiment, an aromatic-cationic peptide is administered to asubject to treat or prevent retinal degeneration. Retinal degenerationis a genetic disease that relates to the break-down of the retina.Retinal tissue may degenerate for various reasons, such as, artery orvein occlusion, diabetic retinopathy, retinopathy of prematurity, and/orretrolental fibroplasia. Retinal degradation generally includesretinoschisis, lattic degeneration, and is related to progressivemacular degeneration. The symptoms of retina degradation include, butare not limited to, impaired vision, loss of vision, night blindness,tunnel vision, loss of peripheral vision, retinal detachment, and/orlight sensitivity.

In one embodiment, an aromatic-cationic peptide is administered to asubject to treat or prevent oxygen-induced retinopathy. Oxygen-inducedretinopathy (OIR) is a disease characterized by microvasculardegeneration. OIR is an established model for studying retinopathy ofprematurity. OIR is associated with vascular cell damage that culminatesin abnormal neovascularization. Microvascular degeneration leads toischemia which contributes to the physical changes associated with OIR.Oxidative stress also plays an important role in the vasoobliteration ofOIR where endothelial cells are prone to peroxidative damage. Pericytes,smooth muscle cells, and perivascular astrocytes, however, are generallyresistant to peroxidative injury. See, e.g., Beauchamp et al., Role ofthromboxane in retinal microvascular degeneration in oxygen-inducedretinopathy, J Appl Physiol. 90: 2279-2288 (2001). OIR, includingretinopathy of prematurity, is generally asymptomatic. However, abnormaleye movements, crossed eyes, severe nearsightedness, and/or leukocoria,can be a sign of OIR or retinopathy of prematurity.

In one aspect, the invention provides a method for preventing, in asubject, an ophthalmic condition by administering to the subject anaromatic-cationic peptide that modulates one or more signs or markers ofan ophthalmic condition. Subjects at risk for an ophthalmic conditioncan be identified by, e.g., any or a combination of diagnostic orprognostic assays as described herein. In prophylactic applications,pharmaceutical compositions or medicaments of aromatic-cationic peptidesare administered to a subject susceptible to, or otherwise at risk of adisease or condition in an amount sufficient to eliminate or reduce therisk, lessen the severity, or delay the outset of the disease, includingbiochemical, histologic and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. Administration of a prophylacticaromatic-cationic can occur prior to the manifestation of symptomscharacteristic of the aberrancy, such that a disease or disorder isprevented or, alternatively, delayed in its progression. Depending uponthe type of aberrancy, e.g., an aromatic-cationic peptide which acts toenhance or improve mitochondrial function or reduce oxidative damage canbe used for treating the subject. The appropriate compound can bedetermined based on screening assays described herein.

Determination of the Biological Effect of the Aromatic-CationicPeptide-Based Therapeutic.

In various embodiments, suitable in vitro or in vivo assays areperformed to determine the effect of a specific aromatic-cationicpeptide-based therapeutic and whether its administration is indicatedfor treatment. In various embodiments, in vitro assays can be performedwith representative cells of the type(s) involved in the subject'sdisorder, to determine if a given aromatic-cationic peptide-basedtherapeutic exerts the desired effect upon the cell type(s). Compoundsfor use in therapy can be tested in suitable animal model systemsincluding, but not limited to rats, mice, chicken, cows, monkeys,rabbits, and the like, prior to testing in human subjects. Similarly,for in vivo testing, any of the animal model system known in the art canbe used prior to administration to human subjects. In one embodiment,administration of an aromatic-cationic peptide to a subject exhibitingsymptoms associated with an ophthalmic condition will cause animprovement in one or more of those symptoms.

Modes of Administration and Effective Dosages

Any method known to those in the art for contacting a cell, organ ortissue with a peptide may be employed. Suitable methods include invitro, ex vivo, or in vivo methods. In vivo methods typically includethe administration of an aromatic-cationic peptide, such as thosedescribed above, to a mammal, preferably a human. When used in vivo fortherapy, the aromatic-cationic peptides are administered to the subjectin effective amounts (i.e., amounts that have desired therapeuticeffect). The dose and dosage regimen will depend upon the degree of theophthalmic condition in the subject, the characteristics of theparticular aromatic-cationic peptide used, e.g., its therapeutic index,the subject, and the subject's history.

The effective amount may be determined during pre-clinical trials andclinical trials by methods familiar to physicians and clinicians. Aneffective amount of a peptide useful in the methods of the presentinvention, preferably in a pharmaceutical composition, may beadministered to a mammal in need thereof by any of a number ofwell-known methods for administering pharmaceutical compounds. In someembodiments, the peptide may be administered systemically, topically, orintraocularly.

The aromatic-cationic peptides described herein can be incorporated intopharmaceutical compositions for administration, singly or incombination, to a subject for the treatment or prevention of a disorderdescribed herein. Such compositions typically include the active agentand a pharmaceutically acceptable carrier. As used herein the term“pharmaceutically acceptable carrier” includes saline, solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions.

Pharmaceutical compositions are typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include parenteral (e.g., intravenous, intradermal,intraperitoneal or subcutaneous), oral, inhalation, transdermal(topical), intraocular, iontophoretic, and transmucosal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. For convenience of thepatient or treating physician, the dosing formulation can be provided ina kit containing all necessary equipment (e.g., vials of drug, vials ofdiluent, syringes and needles) for a treatment course.

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, a composition for parenteral administration must be sterile andshould be fluid to the extent that easy syringability exists. It shouldbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

The aromatic-cationic peptide compositions can include a carrier, whichcan be a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prevention of theaction of microorganisms can be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thiomerasol, and the like. Glutathione and otherantioxidants can be included to prevent oxidation. In many cases, it maybe desirable to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, typical methods of preparation includevacuum drying and freeze drying, which can yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

For ophthalmic applications, the therapeutic compound is formulated intosolutions, suspensions, and ointments appropriate for use in the eye.For ophthalmic formulations generally, see Mitra (ed.), Ophthalmic DrugDelivery Systems, Marcel Dekker, Inc., New York, N.Y. (1993) and alsoHavener, W. H., Ocular Pharmacology, C.V. Mosby Co., St. Louis (1983).Ophthalmic pharmaceutical compositions may be adapted for topicaladministration to the eye in the form of solutions, suspensions,ointments, creams or as a solid insert. For a single dose, from between0.1 ng to 5000 μg, 1 ng to 500 μg, or 10 ng to 100 μg of thearomatic-cationic peptides can be applied to the human eye.

The ophthalmic preparation may contain non-toxic auxiliary substancessuch as antibacterial components which are non-injurious in use, forexample, thimerosal, benzalkonium chloride, methyl and propyl paraben,benzyldodecinium bromide, benzyl alcohol, or phenylethanol; bufferingingredients such as sodium chloride, sodium borate, sodium acetate,sodium citrate, or gluconate buffers; and other conventional ingredientssuch as sorbitan monolaurate, triethanolamine, polyoxyethylene sorbitanmonopalmitylate, ethylenediamine tetraacetic acid, and the like.

The ophthalmic solution or suspension may be administered as often asnecessary to maintain an acceptable level of the aromatic-cationicpeptide in the eye. Administration to the mammalian eye may be aboutonce or twice daily.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Pharmaceuticallycompatible binding agents, and/or adjuvant materials can be included aspart of the composition. The tablets, pills, capsules, troches and thelike can contain any of the following ingredients, or compounds of asimilar nature: a binder such as microcrystalline cellulose, gumtragacanth or gelatin; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, or corn starch; alubricant such as magnesium stearate or Sterotes; a glidant such ascolloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or orange flavoring.

For administration by inhalation, the compounds can be delivered in theform of an aerosol spray from a pressurized container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

Systemic administration of a therapeutic compound as described hereincan also be by transmucosal or transdermal means. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be accomplished through the use of nasalsprays. For transdermal administration, the active compounds areformulated into ointments, salves, gels, or creams as generally known inthe art. In one embodiment, transdermal administration may be performedby iontophoresis.

A therapeutic protein or peptide can be formulated in a carrier system.The carrier can be a colloidal system. The colloidal system can be aliposome, a phospholipid bilayer vehicle. In one embodiment, thetherapeutic peptide is encapsulated in a liposome while maintainingpeptide integrity. As one skilled in the art would appreciate, there area variety of methods to prepare liposomes. (See Lichtenberg et al.,Methods Biochem. Anal., 33:337-462 (1988); Anselem et al., LiposomeTechnology, CRC Press (1993)). Liposomal formulations can delayclearance and increase cellular uptake (See Reddy, Ann. Pharmacother.,34 (7-8):915-923 (2000)). An active agent can also be loaded into aparticle prepared from pharmaceutically acceptable ingredientsincluding, but not limited to, soluble, insoluble, permeable,impermeable, biodegradable or gastroretentive polymers or liposomes.Such particles include, but are not limited to, nanoparticles,biodegradable nanoparticles, microparticles, biodegradablemicroparticles, nano spheres, biodegradable nano spheres, microspheres,biodegradable microspheres, capsules, emulsions, liposomes, micelles andviral vector systems.

The carrier can also be a polymer, e.g., a biodegradable, biocompatiblepolymer matrix. In one embodiment, the therapeutic peptide can beembedded in the polymer matrix, while maintaining protein integrity. Thepolymer may be natural, such as polypeptides, proteins orpolysaccharides, or synthetic, such as poly α-hydroxy acids. Examplesinclude carriers made of, e.g., collagen, fibronectin, elastin,cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin,and combinations thereof. In one embodiment, the polymer is poly-lacticacid (PLA) or copoly lactic/glycolic acid (PGLA). The polymeric matricescan be prepared and isolated in a variety of forms and sizes, includingmicrospheres and nano spheres. Polymer formulations can lead toprolonged duration of therapeutic effect. (See Reddy, Ann.Pharmacother., 34 (7-8):915-923 (2000)). A polymer formulation for humangrowth hormone (hGH) has been used in clinical trials. (See Kozarich andRich, Chemical Biology, 2:548-552 (1998)).

Examples of polymer microsphere sustained release formulations aredescribed in PCT publication WO 99/15154 (Tracy et al.), U.S. Pat. Nos.5,674,534 and 5,716,644 (both to Zale et al.), PCT publication WO96/40073 (Zale et al.), and PCT publication WO 00/38651 (Shah et al.).U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96/40073describe a polymeric matrix containing particles of erythropoietin thatare stabilized against aggregation with a salt.

In some embodiments, the therapeutic compounds are prepared withcarriers that will protect the therapeutic compounds against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylacetic acid. Such formulations can be preparedusing known techniques. The materials can also be obtained commercially,e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomalsuspensions (including liposomes targeted to specific cells withmonoclonal antibodies to cell-specific antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

The therapeutic compounds can also be formulated to enhanceintracellular delivery. For example, liposomal delivery systems areknown in the art, see, e.g., Chonn and Cullis, “Recent Advances inLiposome Drug Delivery Systems,” Current Opinion in Biotechnology6:698-708 (1995); Weiner, “Liposomes for Protein Delivery: SelectingManufacture and Development Processes,” Immunomethods 4 (3) 201-9(1994); and Gregoriadis, “Engineering Liposomes for Drug Delivery:Progress and Problems,” Trends Biotechnol. 13 (12):527-37 (1995).Mizguchi et al., Cancer Lett. 100:63-69 (1996), describes the use offusogenic liposomes to deliver a protein to cells both in vivo and invitro.

Dosage, toxicity and therapeutic efficacy of the therapeutic agents canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds which exhibit high therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the methods, the therapeutically effective dose can be estimatedinitially from cell culture assays. A dose can be formulated in animalmodels to achieve a circulating plasma concentration range that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

Typically, an effective amount of the aromatic-cationic peptides,sufficient for achieving a therapeutic or prophylactic effect, rangefrom about 0.000001 mg per kilogram body weight per day to about 10,000mg per kilogram body weight per day. Preferably, the dosage ranges arefrom about 0.0001 mg per kilogram body weight per day to about 100 mgper kilogram body weight per day. For example dosages can be 1 mg/kgbody weight or 10 mg/kg body weight every day, every two days or everythree days or within the range of 1-10 mg/kg every week, every two weeksor every three weeks. In one embodiment, a single dosage of peptideranges from 0.1-10,000 micrograms per kg body weight. In one embodiment,aromatic-cationic peptide concentrations in a carrier range from 0.2 to2000 micrograms per delivered milliliter. An exemplary treatment regimeentails administration once per day or once a week. Intervals can alsobe irregular as indicated by measuring blood levels of glucose orinsulin in the subject and adjusting dosage or administrationaccordingly. In therapeutic applications, a relatively high dosage atrelatively short intervals is sometimes required until progression ofthe disease is reduced or terminated, and preferably until the subjectshows partial or complete amelioration of symptoms of disease.Thereafter, the patient can be administered a prophylactic regime.

In some embodiments, a therapeutically effective amount of anaromatic-cationic peptide may be defined as a concentration of peptideat the target tissue of 10⁻¹¹ to 10⁻⁶ molar, e.g., approximately 10⁻⁷molar. This concentration may be delivered by systemic doses of 0.001 to100 mg/kg or equivalent dose by body surface area. The schedule of doseswould be optimized to maintain the therapeutic concentration at thetarget tissue, most preferably by single daily or weekly administration,but also including continuous administration (e.g., parenteral infusionor transdermal application).

In some embodiments, the dosage of the aromatic-cationic peptide isprovided at a “low,” “mid,” or “high” dose level. In one embodiment, thelow dose is provided from about 0.0001 to about 0.5 mg/kg/h, suitablyfrom about 0.01 to about 0.1 mg/kg/h. In one embodiment, the mid-dose isprovided from about 0.1 to about 1.0 mg/kg/h, suitably from about 0.1 toabout 0.5 mg/kg/h. In one embodiment, the high dose is provided fromabout 0.5 to about 10 mg/kg/h, suitably from about 0.5 to about 2mg/kg/h.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to, the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of the therapeutic compositionsdescribed herein can include a single treatment or a series oftreatments.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to, the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of the therapeutic compositionsdescribed herein can include a single treatment or a series oftreatments.

The mammal treated in accordance present methods can be any mammal,including, for example, farm animals, such as sheep, pigs, cows, andhorses; pet animals, such as dogs and cats; laboratory animals, such asrats, mice and rabbits. In a preferred embodiment, the mammal is ahuman.

Combination Therapy with an Aromatic-Cationic Peptide and OtherTherapeutic Agents

In certain instances, it may be appropriate to administer at least oneof the aromatic-cationic peptides described herein (or apharmaceutically acceptable salt, ester, amide, prodrug, or solvate) incombination with another therapeutic agent. By way of example only, ifone of the side effects experienced by a patient upon receiving one ofthe aromatic-cationic peptides herein is inflammation, then it may beappropriate to administer an anti-inflammatory agent in combination withthe initial therapeutic agent. Or, by way of example only, thetherapeutic effectiveness of one of the compounds described herein maybe enhanced by administration of an adjuvant (i.e., by itself theadjuvant may only have minimal therapeutic benefit, but in combinationwith another therapeutic agent, the overall therapeutic benefit to thepatient is enhanced). Or, by way of example only, the benefit ofexperienced by a patient may be increased by administering one of thecompounds described herein with another therapeutic agent (which alsoincludes a therapeutic regimen) that also has therapeutic benefit in theprevention or treatment of ophthalmic conditions. By way of exampleonly, in a treatment for macular degeneration involving administrationof one of the aromatic-cationic peptides described herein, increasedtherapeutic benefit may result by also providing the patient with othertherapeutic agents or therapies for macular degeneration. In any case,regardless of the ophthalmic disease, disorder or condition beingtreated, the overall benefit experienced by the patient may simply beadditive of the two therapeutic agents or the patient may experience asynergistic benefit.

Specific, non-limiting examples of possible combination therapiesinclude use of at least one aromatic-cationic peptide with nitric oxide(NO) inducers, statins, negatively charged phospholipids, antioxidants,minerals, anti-inflammatory agents, anti-angiogenic agents, matrixmetalloproteinase inhibitors, and carotenoids. In several instances,suitable combination agents may fall within multiple categories (by wayof example only, lutein is an antioxidant and a carotenoid). Further,the aromatic-cationic peptides may also be administered with additionalagents that may provide benefit to the patient, including by way ofexample only cyclosporin A.

In addition, the aromatic-cationic peptides may also be used incombination with procedures that may provide additional or synergisticbenefit to the patient, including, by way of example only, the use ofextracorporeal rheopheresis (also known as membrane differentialfiltration), the use of implantable miniature telescopes, laserphotocoagulation of drusen, and micro stimulation therapy.

The use of antioxidants has been shown to benefit patients with maculardegenerations and dystrophies. See, e.g., Arch. Ophthalmol., 119:1417-36 (2001); Sparrow, et al., J. Biol. Chem., 278:18207-13 (2003).Examples of suitable antioxidants that could be used in combination withat least one aromatic-cationic peptide include vitamin C, vitamin E,beta-carotene and other carotenoids, coenzyme Q,4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (also known as Tempol),lutein, butylated hydroxytoluene, resveratrol, a trolox analogue(PNU-83836-E), and bilberry extract.

The use of certain minerals has also been shown to benefit patients withmacular degenerations and dystrophies. See, e.g., Arch. Ophthalmol.,119: 1417-36 (2001). Examples of suitable minerals that could be used incombination with at least one aromatic-cationic peptide includecopper-containing minerals, such as cupric oxide; zinc-containingminerals, such as zinc oxide; and selenium-containing compounds.

The use of certain negatively-charged phospholipids has also been shownto benefit patients with macular degenerations and dystrophies. See,e.g., Shaban & Richter, Biol. Chem., 383:537-45 (2002); Shaban, et al.,Exp. Eye Res., 75:99-108 (2002). Examples of suitable negatively chargedphospholipids that could be used in combination with at least onearomatic-cationic peptide include cardiolipin and phosphatidylglycerol.Positively-charged and/or neutral phospholipids may also provide benefitfor patients with macular degenerations and dystrophies when used incombination with aromatic-cationic peptides.

The use of certain carotenoids has been correlated with the maintenanceof photoprotection necessary in photoreceptor cells. Carotenoids arenaturally-occurring yellow to red pigments of the terpenoid group thatcan be found in plants, algae, bacteria, and certain animals, such asbirds and shellfish. Carotenoids are a large class of molecules in whichmore than 600 naturally occurring carotenoids have been identified.Carotenoids include hydrocarbons (carotenes) and their oxygenated,alcoholic derivatives (xanthophylls). They include actinioerythrol,astaxanthin, canthaxanthin, capsanthin, capsorubin, β-8′-apo-carotenal(apo-carotenal), β-12′-apo-carotenal, α-carotene, β-carotene, “carotene”(a mixture of α- and β-carotenes), γ-carotenes, β-cyrptoxanthin, lutein,lycopene, violerythrin, zeaxanthin, and esters of hydroxyl- orcarboxyl-containing members thereof. Many of the carotenoids occur innature as cis- and trans-isomeric forms, while synthetic compounds arefrequently racemic mixtures.

In humans, the retina selectively accumulates mainly two carotenoids:zeaxanthin and lutein. These two carotenoids are thought to aid inprotecting the retina because they are powerful antioxidants and absorbblue light. Studies with quails establish that groups raised oncarotenoid-deficient diets had retinas with low concentrations ofzeaxanthin and suffered severe light damage, as evidenced by a very highnumber of apoptotic photoreceptor cells, while the group with highzeaxanthin concentrations had minimal damage. Examples of suitablecarotenoids for in combination with at least one aromatic-cationicpeptide include lutein and zeaxanthin, as well as any of theaforementioned carotenoids.

Suitable nitric oxide inducers include compounds that stimulateendogenous NO or elevate levels of endogenous endothelium-derivedrelaxing factor (EDRF) in vivo or are substrates for nitric oxidesynthase. Such compounds include, for example, L-arginine,L-homoarginine, and N-hydroxy-L-arginine, including their nitrosated andnitrosylated analogs (e.g., nitrosated L-arginine, nitrosylatedL-arginine, nitrosated N-hydroxy-L-arginine, nitrosylatedN-hydroxy-L-arginine, nitrosated L-homoarginine and nitrosylatedL-homoarginine), precursors of L-arginine and/or physiologicallyacceptable salts thereof, including, for example, citrulline, ornithine,glutamine, lysine, polypeptides comprising at least one of these aminoacids, inhibitors of the enzyme arginase (e.g., N-hydroxy-L-arginine and2(S)-amino-6-boronohexanoic acid) and the substrates for nitric oxidesynthase, cytokines, adenosine, bradykinin, calreticulin, bisacodyl, andphenolphthalein. EDRF is a vascular relaxing factor secreted by theendothelium, and has been identified as nitric oxide or a closelyrelated derivative thereof (Palmer et al, Nature, 327:524-526 (1987);Ignarro et al, Proc. Natl. Acad. Sci. USA, 84:9265-9269 (1987)).

Statins serve as lipid-lowering agents and/or suitable nitric oxideinducers. In addition, a relationship has been demonstrated betweenstatin use and delayed onset or development of macular degeneration. G.McGwin, et al., British Journal of Ophthalmology, 87:1121-25 (2003).Statins can thus provide benefit to a patient suffering from anophthalmic condition (such as the macular degenerations and dystrophies,and the retinal dystrophies) when administered in combination witharomatic-cationic peptides. Suitable statins include, by way of exampleonly, rosuvastatin, pitivastatin, simvastatin, pravastatin,cerivastatin, mevastatin, velostatin, fluvastatin, compactin,lovastatin, dalvastatin, fluindostatin, atorvastatin, atorvastatincalcium (which is the hemicalcium salt of atorvastatin), anddihydrocompactin.

Suitable anti-inflammatory agents with which the aromatic-cationicpeptides may be used include, by way of example only, aspirin and othersalicylates, cromolyn, nedocromil, theophylline, zileuton, zafirlukast,montelukast, pranlukast, indomethacin, and lipoxygenase inhibitors;non-steroidal antiinflammatory drugs (NSAIDs) (such as ibuprofen andnaproxin); prednisone, dexamethasone, cyclooxygenase inhibitors (i.e.,COX-1 and/or COX-2 inhibitors such as Naproxen™, or Celebrex™); statins(by way of example only, rosuvastatin, pitivastatin, simvastatin,pravastatin, cerivastatin, mevastatin, velostatin, fluvastatin,compactin, lovastatin, dalvastatin, fluindostatin, atorvastatin,atorvastatin calcium (which is the hemicalcium salt of atorvastatin),and dihydrocompactin); and disassociated steroids.

Suitable matrix metalloproteinases (MMPs) inhibitors may also beadministered in combination with aromatic-cationic peptides in order totreat ophthalmic conditions or symptoms associated with macular orretinal degenerations. MMPs are known to hydrolyze most components ofthe extracellular matrix. These proteinases play a central role in manybiological processes such as normal tissue remodeling, embryogenesis,wound healing and angiogenesis. However, excessive expression of MMP hasbeen observed in many disease states, including macular degeneration.Many MMPs have been identified, most of which are multidomain zincendopeptidases. A number of metalloproteinase inhibitors are known (seefor example the review of MMP inhibitors by Whittaker M. et al, ChemicalReviews 99(9):2735-2776 (1999)). Representative examples of MMPInhibitors include Tissue Inhibitors of Metalloproteinases (TIMPs)(e.g., TIMP-1, TIMP-2, TIMP-3, or TIMP-4), α-2-macroglobulin,tetracyclines (e.g., tetracycline, minocycline, and doxycycline),hydroxamates (e.g., BATIMASTAT, MARIMISTAT and TROCADE), chelators(e.g., EDTA, cysteine, acetylcysteine, D-penicillamine, and gold salts),synthetic MMP fragments, succinyl mercaptopurines, phosphonamidates, andhydroxaminic acids. Examples of MMP inhibitors that may be used incombination with aromatic cationic peptides include, by way of exampleonly, any of the aforementioned inhibitors.

The use of antiangiogenic or anti-VEGF drugs has also been shown toprovide benefit for patients with macular degenerations and dystrophies.Examples of suitable antiangiogenic or anti-VEGF drugs that could beused in combination with at least one aromatic-cationic peptide includeRhufab V2 (Lucentis™), Tryptophanyl-tRNA synthetase (TrpRS), Eye001(Anti-VEGF Pegylated Aptamer), squalamine, Retaane™ 15 mg (anecortaveacetate for depot suspension; Alcon, Inc.), Combretastatin A4 Prodrug(CA4P), Macugen™, Mifeprex™ (mifepristone—ru486), subtenon triamcinoloneacetonide, intravitreal crystalline triamcinolone acetonide, Prinomastat(AG3340—synthetic matrix metalloproteinase inhibitor, Pfizer),fluocinolone acetonide (including fluocinolone intraocular implant,Bausch & Lomb/Control Delivery Systems), VEGFR inhibitors (Sugen), andVEGF-Trap (Regeneron/Aventis).

Other pharmaceutical therapies that have been used to relieve visualimpairment can be used in combination with at least onearomatic-cationic peptide. Such treatments include but are not limitedto agents such as Visudyne™ with use of a non-thermal laser, PKC 412,Endovion (NeuroSearch A/S), neurotrophic factors, including by way ofexample Glial Derived Neurotrophic Factor and Ciliary NeurotrophicFactor, diatazem, dorzolamide, Phototrop, 9-cis-retinal, eye medication(including Echo Therapy) including phospholine iodide or echothiophateor carbonic anhydrase inhibitors, AE-941 (AEterna Laboratories, Inc.),Sirna-027 (Sirna Therapeutics, Inc.), pegaptanib (NeXstarPharmaceuticals/Gilead Sciences), neurotrophins (including, by way ofexample only, NT-4/5, Genentech), Candy (Acuity Pharmaceuticals),ranibizumab (Genentech), INS-37217 (Inspire Pharmaceuticals), integrinantagonists (including those from Jerini AG and Abbott Laboratories),EG-3306 (Ark Therapeutics Ltd.), BDM-E (BioDiem Ltd.), thalidomide (asused, for example, by EntreMed, Inc.), cardiotrophin-1 (Genentech),2-methoxyestradiol (Allergan/Oculex), DL-8234 (Toray Industries),NTC-200 (Neurotech), tetrathiomolybdate (University of Michigan),LYN-002 (Lynkeus Biotech), microalgal compound (Aquasearch/Albany, MeraPharmaceuticals), D-9120 (Celltech Group plc), ATX-S10 (HamamatsuPhotonics), TGF-beta 2 (Genzyme/Celtrix), tyrosine kinase inhibitors(Allergan, SUGEN, Pfizer), NX-278-L (NeXstar Pharmaceuticals/GileadSciences), Opt-24 (OPTIS France SA), retinal cell ganglionneuroprotectants (Cogent Neurosciences), N-nitropyrazole derivatives(Texas A&M University System), KP-102 (Krenitsky Pharmaceuticals), andcyclosporin A.

In any case, the multiple therapeutic agents may be administered in anyorder or even simultaneously. If simultaneously, the multipletherapeutic agents may be provided in a single, unified form, or inmultiple forms (by way of example only, either as a single solution oras two separate solutions). One of the therapeutic agents may be givenin multiple doses, or both may be given as multiple doses. If notsimultaneous, the timing between the multiple doses may vary from morethan zero weeks to less than about four weeks, less than about sixweeks, less than about 2 months, less than about 4 months, less thanabout 6 months, or less than about one year. In addition, thecombination methods, compositions and formulations are not to be limitedto the use of only two agents. By way of example only, anaromatic-cationic peptide may be provided with at least one antioxidantand at least one negatively charged phospholipid; or anaromatic-cationic peptide may be provided with at least one antioxidantand at least one inducer of nitric oxide production; or anaromatic-cationic peptide may be provided with at least one inducer ofnitric oxide productions and at least one negatively chargedphospholipid; and so forth.

In addition, an aromatic-cationic peptide may also be used incombination with procedures that may provide additional or synergisticbenefits to the patient. Procedures known, proposed or considered torelieve visual impairment include but are not limited to “limitedretinal translocation”, photodynamic therapy (including, by way ofexample only, receptor-targeted PDT, Bristol-Myers Squibb, Co.; porfimersodium for injection with PDT; verteporfin, QLT Inc.; rostaporfin withPDT, Miravent Medical Technologies; talaporfin sodium with PDT, NipponPetroleum; motexafin lutetium, Pharmacyclics, Inc.), antisenseoligonucleotides (including, by way of example, products tested byNovagali Pharma SA and ISIS-13650, Isis Pharmaceuticals), laserphotocoagulation, drusen lasering, macular hole surgery, maculartranslocation surgery, implantable miniature telescopes, Phi-MotionAngiography (also known as Micro-Laser Therapy and Feeder VesselTreatment), Proton Beam Therapy, microstimulation therapy, RetinalDetachment and Vitreous Surgery, Scleral Buckle, Submacular Surgery,Transpupillary Thermotherapy, Photosystem I therapy, use of RNAinterference (RNAi), extracorporeal rheopheresis (also known as membranedifferential filtration and Rheotherapy), microchip implantation, stemcell therapy, gene replacement therapy, ribozyme gene therapy (includinggene therapy for hypoxia response element, Oxford Biomedica; Lentipak,Genetix; PDEF gene therapy, GenVec), photoreceptor/retinal cellstransplantation (including transplantable retinal epithelial cells,Diacrin, Inc.; retinal cell transplant, Cell Genesys, Inc.), andacupuncture.

Further combinations that may be used to benefit an individual includeusing genetic testing to determine whether that individual is a carrierof a mutant gene that is known to be correlated with certain ophthalmicconditions. By way of example only, defects in the human ABCA4 gene arethought to be associated with five distinct retinal phenotypes includingStargardt disease, cone-rod dystrophy, age-related macular degenerationand retinitis pigmentosa. See e.g., Allikmets et al., Science,277:1805-07 (1997); Lewis et al., Am. J. Hum. Genet., 64:422-34 (1999);Stone et al., Nature Genetics, 20:328-29 (1998); Allikmets, Am. J Hum.Gen., 67:793-799 (2000); Klevering, et al., Ophthalmology, 11 1:546-553(2004). In addition, an autosomal dominant form of Stargardt Disease iscaused by mutations in the ELOV4 gene. See Karan, et al., Proc. Natl.Acad. Sci. (2005). Patients possessing any of these mutations areexpected to find therapeutic and/or prophylactic benefit in the methodsdescribed herein.

EXAMPLES

The present invention is further illustrated by the following examples,which should not be construed as limiting in any way.

Example 1—Prevention of High Glucose Induced Injury of Human RetinalEpithelial Cells

The effects of the aromatic-cationic peptides of the invention inpreventing high glucose induced injury in human retinal epithelial cells(HREC) were investigated in cultured HRECs.

Methods of HREC culture useful in the studies of the present inventionare known. See generally, Li B, Tang S B, Zhang G, Chen J H, Li B J.Culture and characterization of human retinal capillary endothelialcell. Chin Ophthal Res 2005; 23: 20-2; Premanand C, Rema M, Sameer M Z,Sujatha M, Balasubramanyam M. Effect of curcumin on proliferation ofhuman retinal endothelial cells under in vitro conditions. InvestOphthalmol Vis Sci 2006; 47: 2179-84.

Briefly, HREC cells were divided into three groups: a normal controlgroup; a group administered 30 mM glucose; and a group administered 30mM glucose+SS-31. Survival of HRECs in high glucose co-treated withdifferent concentrations of SS-31 (10 nM, 100 nM, 1 μM, 10 μM) wasmeasured using an Annexin V+PI assay and flow cytometry. See generally,Koopman, G., Reutelingsperger, C. P., Kuijten, G. A. M., Keehnen, R. M.J., Pals, S. T., and van Oers, M. H. J. 1994. Annexin V for flowcytometric detection of phosphatidylserine expression on B cellsundergoing apoptosis. Blood 84: 1415; Homburg, C. H., de Haas, M., vondem Borne, A. E., Verhoeven, A. J., Reutelingsperger, C. P., and Roos,D. 1995. Human neutrophils lose their surface Fc gamma RIII and acquireAnnexin V binding sites during apoptosis in vitro. Blood 85: 532;Vermes, I., Haanen, C., Steffens-Nakken, H., and Reutelingsperger, C.1995. A novel assay for apoptosis—flow cytometric detection ofphosphatidylserine expression on early apoptotic cells using fluoresceinlabelled Annexin V. J. Immunol. Meth. 184: 39; Fadok, V. A., Voelker, D.R., Campbell, P. A., Cohen, J. J., Bratton, D. L., and Henson, P. M.1992. Exposure of phosphatidylserine on the surface of apoptoticlymphocytes triggers specific recognition and removal by macrophages. J.Immunol. 148: 2207.

The survival of HRECs in high glucose co-treated with SS-31 was testedat 24 h and 48 h. The results are shown in FIG. 1 and indicate thatsurvival of HRECs was significantly improved with the administration ofSS-31, with a reduction in apoptotic and necrotic cells. The treatmentof SS-31 also reduced the production of ROS (FIG. 2).

Assessment of SS-31 as a protectant against mitochondrial potential lossof HRECs treated with high-glucose was examined. To determine if amitochondrial-mediated pathway was important in SS31's protective effectagainst high glucose-induced cell death, ΔΨm was measured by flowcytometry. After treating the HRECs with high-glucose without SS31 for24 or 48 hours, a rapid loss of mitochondrial membrane potential wasdetected by JC-1 fluorescent probe as indicated by a significantdecrease in the ratio of red to green fluorescence observed in the highglucose group. In contrast, ΔΨm in the 100 nM SS31 co-treated groupremained virtually unchanged and was comparable to the normal glucosecontrol group (FIG. 3). These data suggest that SS31 prevented themitochondrial membrane potential loss caused by exposure to a highglucose environment.

Glucose (30 mmol/L) induced cytochrome c release from the mitochondriaof HRECs. Fixed HRECs were immunolabeled with a cytochrome c antibodyand a mitochondrial specific protein antibody (HSP60). Confocalmicroscopic analysis showed that HRECs in normal culture and in SS-31co-treated with glucose have overlapping cytochrome c staining andmitochondria staining, indicating colocalization of cytochrome c andmitochondria (FIG. 4). After treatment with 30 mmol/L glucose for 24 hor 48 h, some cytochrome c was observed in the cytoplasm of HRECs,indicating that glucose induces the release of cytochrome c from themitochondria to cytoplasm in HREC cells, but SS-31 can decrease suchtranslocation between mitochondria and cytoplasm.

The prevention of cytochrome c release from mitochondria resulted inreduced caspase-3 activity. As shown in FIG. 5, SS-31 decreased theprotein expression of caspase-3 in high glucose-treated HRECs. The levelof cleaved caspase-3 protein expression was measured by Western blot(FIG. 5A). When HRECs were exposed to 30 mM glucose for 24 h and 48 h,the level of caspase-3 expression increased dramatically. At the sametime, in the SS-31 co-treated group, it displayed a marked decrease inthe caspase-3 protein level (*p<0.05). FIG. 5B shows a quantitativeanalysis the level of caspase-3 expression of HRECs in high glucoseco-treated with SS-31 for 24 and 48 h.

SS-31 increased the expression of Trx2 in the high glucose-treatedHRECs. FIG. 5C shows the mRNA level of Trx2 in HRECs exposed to 30 mMglucose co-treated with SS-31 for 24 h and 48 h. The mRNA expressionlevel of Trx2 was measured by quantitative real-time PCR. Relative mRNAlevels of Trx2 were normalized by 18S mRNA levels (* p<0.05 vs. thenormal glucose medium group and the 30 mM high glucose treated group).Three independent samples were used for each time point. FIG. 5D showsthe level of Trx2 protein expression as measured by Western blot. Theprotein expression of Trx2 in the high glucose co-treated with SS-31group significantly increased comparing to the normal glucose group(*p<0.05). FIG. 5E shows quantitative analysis of the protein level ofTrx2 in HRECs 24 and 48 h after high glucose without or with SS-31co-treatment.

These results indicate that SS-31 can promote the survival of HREC cellsin a high glucose environment. As such, SS-31 and otheraromatic-cationic peptides may be useful in methods for the preventionof diabetic retinopathy.

Example 2—Prevention of Diabetic Retinopathy in Rats Fed a High Fat Diet

The effects of the aromatic-cationic peptides of the invention inpreventing the development of diabetic retinopathy were investigated ina Sprague-Dawley rat model. The example describes the results of suchexperiments.

A rat model of diabetes was established by combination of 6-week HFD andlow dose of STZ (30 mg/kg) injection or a single high dose of STZ (65mg/kg) in SD rats. See generally, K. Srinivasan, B. Viswanad, LydiaAsrat, C. L. Kaul and P. Ramarao, Combination of high-fat diet-fed andlow-dose streptozotocin-treated rat: A model for type 2 diabetes andpharmacological screening, Pharmacological Research, 52(4): 313-320,2005. Rats of the same batch fed with normal chow (NRC) were used as acontrol. Tables 7-10 show the therapeutic schedule and experimentalprotocol.

TABLE 7 Treatment Groups - HFD/STZ Model Number of Dosage and Group RatsModel Treatment Route A 12 HFD/STZ SS-31 10 mg/kg s.c B 12 HFD/STZ SS-313 mg/kg s.c. C 12 HFD/STZ SS-31 1 mg/kg s.c. D 10 HFD/STZ SS-20 10 mg/kgs.c. E 10 HFD/STZ SS-20 3 mg/kg s.c. F 10 HFD/STZ Saline Equal vol. s.c.G 10 NRC Saline Equal vol. s.c.

TABLE 8 Therapeutic Schedule - HFD/STZ Model Diabetic Groups ControlGroup Duration Objective (A, B, C, D, E, F) (G) 1^(st) Week AcclimationNormal rat chow 2^(nd)-7^(th) Week Diet High Fat Diet Normal Rat ChowManipulation End of 7^(th) Week STZ STZ 30 mg/kg, Citrate bufferInjection i.p., once 8^(th)-27^(th) Week Induction of High fat dietuntil Normal rat chow Diabetes 21^(st) week, then switched to normal ratchow 28^(th)-37^(th) Week Peptide Peptide treatment Group G: 2 Treatment(see Table 7) mL/kg, s.c. 38^(th) Week Collected 24 h urine and bloodsamples, and harvested vital organs

TABLE 9 Treatment Groups -STZ Model Number of Dosage and Group RatsModel Treatment Route A 11 Diabetes SS-31 10 mg/kg s.c B 11 DiabetesSS-20 10 mg/kg s.c. C 10 Diabetes Saline Equal vol. s.c. D 10 NormalSaline Equal vol. s.c.

TABLE 10 Therapeutic Schedule -STZ Model Diabetic Groups Control GroupDuration Objective (A, B, C) (D) 1^(st)-3^(rd) Week Acclimation Normalrat chow End of 3^(rd) Week STZ STZ 30 mg/kg, Citrate buffer Injectioni.p., once 4^(th)-18^(th) Week Induction of Normal Rat Chow DiabeticComplications 19^(h)-28^(th) Week Peptide Peptide treatment Group D: 2Treatment (see Table 9) mL/kg, s.c. 29^(th) Week Collected 24 h urineand blood samples, and harvested vital organs

In accordance with the experimental protocol just described, the effectsof the aromatic-cationic peptides in treating conditions associated withdiabetes in a SD rat model were demonstrated. Administration of SS-20and SS-31 resulted in a prevention or reversal of cataract formation inthe lenses of diabetic rats (FIGS. 6 and 7, Tables 11 and 12).

TABLE 11 HFD/STZ Rat Model Percentage Percentage of Turbidity degree ofopacity severe Group − + ++ +++ ++++ (%) opacity (%) NRC 4 0 0 0 0 0  0 HFD/STZ 1 0 2 3 0 83.3 0  SS20 3 mg 1 1 1 0 1 75.0 25.0 SS20 10 mg 1 2 10 0 75.0 0  SS31 1 mg 1 1 1 0 0 67.7 0  SS31 3 mg 3 1 0 0 1 20.0 20.0SS31 10 mg 6 1 0 0 0 14.3 0  −: transparent; +: mildly opaque; ++:opaque; +++: moderately opaque; ++++: severely opaque

TABLE 12 STZ Rat Model Percentage Percentage of Opacity degree ofopacity severe Group − + ++ +++ ++++ (%) opacity (%) NRC 6 0 0 0 0 0  0 STZ 1 0 0 1 3 80.0 60.0 SS20 10 mg 2 0 2 0 1 60.0 20.0 SS31 10 mg 2 2 00 1 60.0 20.0

The effect of the aromatic cationic peptides on lens epithelium in theSD rat model was investigated. Administration of SS-31 reducedepithelial cellular changes in both STZ rat model (FIG. 8) and HFD/STZrat model (FIG. 9).

The effect of the aromatic-cationic peptides on the inner blood-retinalbarrier function in the SD rat model was investigated. Administration ofSS-20 and SS-31 resulted in improved inner blood-retinal barrierfunction compared to rats on a HFD not administered SS-20 or SS-31 (FIG.10).

The effect of the aromatic-cationic peptides on retinal microvessels inthe SD rat model was investigated (FIGS. 11-12). Administration of SS-31reduced retinal microvascular changes observed in STZ or HFD/STZ rats.

The effect of the aromatic-cationic peptides on the distribution oftight junction protein claudin-5 in retinal microvessels in the SD ratmodel was investigated. Distribution of tight junction protein claudin-5was detected under a confocal microscope (FIG. 13). Claudin-5 wasdistributed along the retinal vessels smoothly, linearly, and uniformlyin normal rats (A), but the linear shape was broken in the STZ rat (B,arrow). Distribution of claudin-5 on retinal vessels in STZ rats treatedwith SS-20 (10 mg/kg) or SS-31 (10 mg/kg) was similar to that of normalrat (Panels C and D, respectively).

In summary, these findings collectively establish that aromatic-cationicpeptides, either prevent or compensate for the negative effects ofdiabetes in the eye, e.g., cataracts and microvasculature. As such,administration of the aromatic-cationic peptides of the presentinvention is useful in methods of preventing or treating ophthalmicconditions associated with diabetes in human subjects.

Example 3—SS-31 Prevents Oxidative Stress in Glaucomatous TrabecularMeshwork Cells

The effects of the aromatic-cationic peptides of the invention inpreventing or treating glaucoma were investigated by studying theeffects of the peptides in glaucomatous trabecular meshwork cells.Glaucoma is the second leading cause of irreversible blindnessworldwide. Primary open-angle glaucoma (POAG) is the major subtype ofglaucoma. In POAG, there is no visible abnormality of the trabecularmeshwork. However, it is believed that the ability of the cells in thetrabecular meshwork to carry out their normal function is impaired.

In this Example, the effects of the aromatic-cationic peptides of theinvention were compared between trabecular meshwork cells from POAGpatients (GTM) and trabecular meshwork cells from non-diseasedindividuals (HTM). Methods useful in the studies of the presentinvention have been described. See generally, He Y, Ge J, Tombran-TinkJ., Mitochondrial defects and dysfunction in calcium regulation inglaucomatous trabecular meshwork cells. Invest Ophthalmol Vis Sci. 2008,49(11):4912-22; He Y, Leung K W, Zhang Y H, Duan S, Zhong X F, Jiang RZ, Peng Z, Tombran-Tink J, Ge J. Mitochondrial complex I defect inducesROS release and degeneration in trabecular meshwork cells of POAGpatients: protection by antioxidants. Invest Ophthalmol Vis Sci. 2008,49(4):1447-58. GTM cells show a significant impairment of mitochondrialmembrane potential compared to HTM cells (FIG. 18).

The cells were divided into three groups: “Group A” cells were exposedto hydrogen peroxide prior to administration of SS-31. “Group B” cellswere exposed to SS-31 prior to administration of hydrogen peroxide.“Group C” cells were administered SS-31 and hydrogen peroxidesimultaneously.

To assess whether SS-31 had cytotoxic effects of HTM or GTM cells,various concentrations of SS-31 were administered to cells and thecytotoxicity was measured using an LDH assay. A LDH cytotoxicity assayis a colorimetric method of assaying cellular cytotoxicity. The assayquantitatively measures the stable, cytosolic, lactate dehydrogenase(LDH) enzyme, which is released from damaged cells. The released LDH ismeasured with a coupled enzymatic reaction that results in theconversion of a tetrazolium salt (iodonitrotetrazolium (INT)) into a redcolor formazan by diaphorase. Methods to detect LDH from cells useful inthe studies of the present invention are known. See generally, Haslam,G. et al. (2005) Anal. Biochem. 336: 187; Tarnawski, A. (2005) Biochem.Biophys. Res. Comm. 333: 207; Round, J. L et al. (2005) J. Exp. Med.201: 419; Bose, C. et al. (2005) Am. J. Physiol. Gastr. L. 289: G926;Chen, A. and Xu, J. (2005) Am. J. Physiol. Gastr. L. 288: G447. The LDHactivity is determined as NADH oxidation or INT reduction over a definedtime period. The results are shown in FIG. 14 and indicate that SS-31does not affect the viability of HTM and GTM cells.

Methods to measure mitochondrial membrane potential using TMRM useful inthe studies of the present invention have been described by AndreaRasola and Massimo Geuna, A flow cytometry assay simultaneously detectsindependent apoptotic parameters, Cytometry 45:151-157, 2001; Mitoprobe™JC-1 Kit for Flow Cytometry, Molecular Probes, Invitrogen, USA. FIG. 16shows the results in GTM cells. Collectively, these results establishthat treatment with SS-31 improves the mitochondrial membrane potentialof cells that were exposed to hydrogen peroxide prior to administrationof SS-31.

Group A.

The mitochondrial membrane potential (Δψm) of HTM and GTM cells wasinvestigated when those cells were exposed to hydrogen peroxide prior toadministration of SS-31. First, the mitochondrial membrane potential wasmeasured using confocal microscopy of cells labeled withtetramethylrhodamine methyl ester (TMRM, 500 nM×30 min) (FIG. 15). Themitochondrial membrane potential was also measured using flow cytometry(FIGS. 16-17) by labeling cells with the mitochondrion-selective probetetramethylrhodamine methyl ester (TMRM, 500 nM×30 min).

Group B.

The morphology of GTM cells was investigated when those cells wereexposed to SS-31 prior to administration of hydrogen peroxide. FIG. 18shows the results of inverted phase contrast microscopy of cellsadministered various concentrations of SS-31. The results indicate thatSS-31 protects cells from hydrogen peroxide-mediated morphologicalchanges in a concentration-dependent and time-dependent manner. That is,hydrogen peroxide mediated cell loss and rounding was diminished incells exposed to SS-31 peptide. The mitochondrial membrane potential(Δψm) of HTM and GTM cells was also investigated when those cells wereexposed to SS-31 prior to administration of hydrogen peroxide. Themitochondrial membrane potential was measured using confocal microscopyof cells labeled with tetramethylrhodamine methyl ester (TMRM, 500 nM×30min) (FIG. 19-21). These results show that pre-treatment with SS-31dose-dependently improves the mitochondrial membrane potential of cellsthat were exposed to hydrogen peroxide. As such, SS-31 provides aprotective effect against oxidative stress in GTM cells.

The effects of SS-31 at mitigating acute oxidative injury in GTM and HTMcells was investigated. FIG. 36 shows the fluorescence intensity of TMRMof GTM and HTM cells using FACS analysis. The percentage of fluorescenceintensity compared to GTM control in H₂O₂, SS-31 10⁻⁶M, SS-31 10⁻⁷M,SS-31 10⁻⁸M were 35.2±2.12%, 56.2±4.04%, 50.3±4.46%, 47.5±2.82%respectively, n=4; the HTM groups were 37.4±0.725%, 57.7±1.80%,50.6±3.06%, 49.4±2.27% respectively, n=4. ** means P<0.01 compared toGTM H₂O₂ group; * means P<0.05 compared to GTM H₂O₂ group; ▴ ▴ ▴ meansP<0.001 compared to HTM H₂O₂ group.

FIG. 37 shows the fluorescence intensity of ROS of GTM and HTM cells incontrol and SS-31-treated groups using FACS analysis. The percentage ofintracellular ROS production compared to GTM control in GTM H₂O₂, SS-3110⁻⁶M, SS-31 10⁻⁷M, SS-31 10⁻⁸M groups were 146.0±2.27%, 84.5±8.75%,102.0±5.69%, 133.0±5.17% respectively (n=3); the HTM groups were153.0±3.46%, 79±2.39%, 91.8±3.49%, 129.0±8.24% respectively (n=4).P<0.001 GTM and HTM H₂O₂ Group compared to control; *** means P<0.001compared to GTM H₂O₂ group; ▴ ▴ ▴ Means P<0.001 compared to HTM H₂O₂group; ▴ ▴ means P<0.01 compared to HTM H₂O₂ group. FIG. 38 shows thatSS-31 reduced the amount of cell apoptosis induced by H₂O₂.

The effects of SS-31 on sustained oxidative injury of GTM and HTM cellswas examined. Cells were pre-treated with 10⁻⁶, 10⁻⁷, 10⁻⁸ M of SS-31for 1 h, and then incubated with 200 μM H₂O₂ for 24 h to investigate theprotective effect of SS-31 in sustained oxidative stress. FIG. 39 andTable 13 shows the effects of SS-31 on ROS production from sustainedoxidative injury of GTM and HTM cells. FIG. 40 and Table 14 shows theMMP change in GTM and HTM cells in each treatment group.

TABLE 13 ROS Production in GTM3 and iHTM cells treated with H₂O₂. SS-31H₂O₂-Ctrl (%) 1 μM (%) 0.1 μM (%) 0.01 μM (%) GTM3 376.80 ± 17.47 47.40± 1.81*** 68.91 ± 8.62*** 133.70 ± 3.24*** iHTM 388.50 ± 5.54  36.91 ±1.47*** 82.89 ± 3.70*** 114.30 ± 3.89***

TABLE 14 MMP Decline in GTM3 and iHTM cells treated with H₂O₂. SS-31H2O2-Ctrl (%) 1 μM (%) 0.1 μM (%) 0.01 μM (%) GTM3 −39.67 ± 2.33 −24.33± 4.18*  −29.33 ± 2.19*  −31.33 ± 1.20* iHTM −69.53 ± 2.01 −44.99 ±2.19*** −53.24 ± 2.52** −58.24 ± 2.62*

Collectively, these results demonstrate that SS-31 has no cytotoxicityat 10⁻⁴M for both GTM and HTM cells and that sustained and acuteoxidative stress induced by hydrogen peroxide can be prevented by SS-31(>10⁻⁹ M). As such, the aromatic-cationic peptides of the presentinvention are useful in methods of preventing or treating glaucoma inhuman subjects.

Example 4—SS-31 Prevents Oxidative Stress in Primary Retinal PigmentEpithelial Cells

Primary retinal pigment epithelial (RPE) cells were cultured to test theeffects of the aromatic-cationic peptides of the invention in preventingor reducing oxidative damage in these cells. Methods useful for thestudy of primary retinal pigment epithelial cells have been described.See, Dunn et al., ARPE-19, A Human Retinal Pigment Epithelial Cell Linewith Differentiated Properties, Experimental Eye Research, 1996, 62(2):155-170. First, it was shown that SS-31 did not adversely effect thesecells. Primary cultured human RPE cells were incubated with differentconcentrations of SS-31 alone for a period of 24 h, and cell viabilitywas determined by a MTT assay (FIG. 22).

Next, the viability of primary RPE cells was tested in the presence oftBHP and various concentrations of SS-31. Cells were plated at 10,000cells per well in a 96-well plate and cultured for 24 h, then starvedfor 24 h. After that, cells were exposed to increasing concentrations oftBHP (FIG. 23A), or preincubated for 4 h with different concentrationsof SS-31, then stimulated with tBHP for 6 h (FIG. 23B). These resultsindicate that SS-31 enhanced cell viability in response to tBHPadministration. Intracellular ROS production in three groups of RPEcells was also examined using FACS analysis. FIG. 31A shows ROSproduction in control RPE cells; FIG. 31B shows ROS production in RPEcells treated with 500 μM tBHP for 3 h; and FIG. 31C shows ROSproduction in RPE cells treated with 500 μM tBHP for 3 h and 1 μM SS-31.FIG. 32 shows MMP labeled by JC-1 in a FACS analysis. Three differentconcentration of SS-31 groups were analyzed. The ratio of red to greenin 500 μM tBHP for the 3 h group is 1.08, the ratio of red to green in10 nM SS-31 for 4 h+500 μM tBHP for 3 h group is 1.25; the ratio of redto green in 100 nM SS-31 for 4 hr+500 μM tBHP for 3 h group is 1.4; andthe ratio of red to green in 1 μM SS-31 for 4 h+500 μM tBHP for 3 hgroup is 2.28. FIG. 33 shows the effect of 1 μM SS-31 on MMP declineinduced by tBHP. FIG. 33A: Control group, R/G is 3.63±0.24; FIG. 33B:500 μM tBHP for 3 h group, R/G is 1.08±0.11; FIG. 33C: 1 μM SS-31 for 4h+500 μM tBHP for 3 h group, R/G is 2.38±0.18. FIG. 33D is a chartcomparing the fluorescence ratio for the different groups. *P<0.01, Cvs. B.

FIG. 34 shows the effect of SS-31 on cell apoptosis induced by 250 μMtBHP for 24 h. FIG. 34A: control group; (Q2+Q4)%=1.27±0.3%; FIG. 34B:250 μM tBHP for 24 h group; (Q2+Q4)%=15.7±0.6%; FIG. 34C: 1 μM SS-31 for4 h+250 μM tBHP for 24 h group; (Q2+Q4)%=8.4±0.8%. FIG. 34D is a chartcomparing the fluorescence ratio for the different groups. *P<0.05 C vs.B. FIG. 35 is a chart showing the MDA level induced by tBHP in 3 groupsof RPE cells. (*P<0.05).

Collectively, these results demonstrate that SS-31 prevents oxidativestress in primary retinal pigment epithelial cells. As such, thearomatic-cationic peptides of the present invention are useful inmethods of preventing or treating damage to retinal cells in humansubjects.

Example 5—Prevention and Treatment of Choroidal Neovascularization byAromatic-Cationic Peptides of the Invention in a CNV Mouse Model

To further demonstrate the prevention of choroidal neovascularization(CNV) on the one hand, and treatment of CNV on the other hand, thearomatic-cationic peptides of the invention were tested on a mouse modelof CNV (FIG. 24). CNV were induced in the eye with laser burns. Methodsuseful in the present studies have been described by Reich, Mol Vis2003; 9:210-216.

Briefly, five to six-week-old C57BL/6 male mice were anesthetized withchloral hydrate and the pupils were dilated with tropicamide. With acoverslip used as a contact lens, four laser spots (532 nm, 260 mw, 0.01s, 50 μm; Novus Spectra, Lumenis, USA) were applied to the fundus in acircle around the optic disc in the right eye. Daily intraperitonealinjections of 1 mg/kg, 9 mg/kg SS-31 or vehicle were started the dayprior to laser photocoagulation.

After one week, mice were deeply anaesthetized and perfused through theleft ventricle with 1 ml (50 mg/ml) of PBS-buffered fluorescein-dextran.Eyes were enucleated and fixed in 4% paraformaldehyde for 2 h. The eyeswere sectioned at the equator, and the anterior half and retina wereremoved. The posterior eye segment containing the sclera and choroid wasdissected into quarters by four to five radial cuts and mounted on aslide. All flatmounts were examined by a fluorescence microscope(AxioCam MRC; Carl Zeiss). Image-Pro Plus software (Media Cybernetics,Silver Spring, Md.) was used to measure the area of each CNV lesion.

There were 48 locations of neovascularization in each group. The area ofneovascularization was calculated using IMAGE-PROPLUS6.0 software. Thearea of neovascularization in the CNV model, 1 mg/kg SS-31 and 9 mg/kgSS-31 groups were 0.0130±0.0034, 0.0068±0.0025, 0.0067±0, respectively.These results indicate that the two concentrations of SS-31significantly reduced the area of choroidal neovasculatization (P<0.05)(FIG. 24).

Example 6—Prevention and Treatment of Oxygen-Induced Retinopathy (OIR)by Aromatic-Cationic Peptides of the Invention in an OIR Mouse Model

To further demonstrate the prevention of oxygen-induced retinopathy(OIR), the aromatic-cationic peptides of the invention were tested on amouse model of OIR (FIG. 25). In this model, 7-day-old mouse pups withpartially developed retinal vasculature were subjected to hyperoxia (75%oxygen) for 5 days, which stops retinal vessel growth and causessignificant vaso-obliteration. On postnatal day 12, the pups werereturned to room air, and by postnatal day 17, a florid compensatoryretinal neovascularization occurred. This model of pathologicalneovascularization has been widely used as a substitute forproliferative diabetic retinopathy (DR).

To examine the effects of the aromatic-cationic peptides of theinvention on prevention of OIR, OIR was induced in mouse pups and themice were simultaneously administered an aromatic-cationic peptide(e.g., SS-20 or SS-31) for approximately 6 weeks. The results are shownin FIG. 26 and indicate that treatment with SS-31 prevented thecompensatory retinal neovascularization. As such, the aromatic-cationicpeptides of the invention are useful in methods of preventingproliferative diabetic retinopathy in mammalian subjects.

Example 7—Antioxidants Reduce Photoreceptor Cell Death in a Model ofRetinitis Pigmentosa

A cone cell specific line 661W was derived from a mouse retinal tumor.Methods useful in the present studies of 661W cells have been describedpreviously. See generally, Gearóid Tuohy, Sophia Millington-Ward, PaulF. Kenna, Peter Humphries and G. Jane Farrar, Sensitivity ofPhotoreceptor-Derived Cell Line (661W) to Baculoviral p35, Z-VAD.FMK,and Fas-Associated Death Domain, Investigative Ophthalmology and VisualScience. 2002; 43:3583-3589. These cells were cultured to test theeffects of the aromatic-cationic peptides of the invention in preventingor reducing oxidative damage in the cone cells (FIG. 27). First, it wasshown that tBHP affected survival of 661W cells (FIG. 27A). Differentdoses of tBHP were administered to the cells for 3 h. Next, it was shownthat different doses of SS-31 reduced tBHP-induced 661W cell death (FIG.27B).

The potential of SS-31 to protect against loss of mitochondrialviability induced by tBHP, 100 nmol/L SS-31 was administered to thecultures of 661w cells. The results are shown in FIG. 30 and indicatethat SS-31 significantly enhanced mitochondrial viability compared tocells not administered SS-31, as shown by a JC-1 assay.

Example 8—Effects of SS-31 in a Mouse Model of Retina Degeneration

To further demonstrate the prevention of retinal degeneration, thearomatic-cationic peptides of the invention were tested on a mouse modelof retina degeneration. CNV is induced in the eye with laser burns. (seeExample 5). Mouse models of retinal degeneration have been investigatedfor many years in the hope of understanding the causes of photoreceptorcell death. Naturally occurring mouse mutants that manifest degenerationof photoreceptors in the retina with preservation of all other retinalcell types have been found: retinal degeneration (formerly rd, identicalwith rodless retina, r, now Pde6b rd1); Purkinje cell degeneration(pcd); nervous (nr); retinal degeneration slow (rds, now Prph Rd2);retinal degeneration 3 (rd3); motor neuron degeneration (mnd); retinaldegeneration 4 (Rd4); retinal degeneration 5 (rd5); vitiligo (vit, nowMitf mi-vit); retinal degeneration 6 (rd6); retinal degeneration 7(rd7); neuronal ceroid lipofuscinosis (nclf); retinal degeneration 8(rd8); retinal degeneration 9 (Rd9); retinal degeneration 10 (rd10); andcone photoreceptor function loss (cpfl1).

FIG. 28 is a series of micrographs showing the thickness of the retinalouter nuclear layer (ONL) in a mouse model of retina degeneration incontrol and SS-31-treated mice. The results indicate that SS-31 treatedmice retained a greater number of rows of cells in the ONL compared tountreated mice. Retinal flat mounts stained with peanut agglutinin(PNA), which selectively stain core inner and outer segments also showthat cone cell density is greater in SS-31 treated mice (FIG. 29). Theseresults indicate that treatment with SS-31 prevented the compensatorydamage to the retinal outer nuclear layer in a mouse model of retinaldegeneration. As such, the aromatic-cationic peptides of the inventionare useful in methods of preventing retinal degeneration in mammaliansubjects.

EQUIVALENTS

The present invention is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the invention. Many modificationsand variations of this invention can be made without departing from itsspirit and scope, as will be apparent to those skilled in the art.Functionally equivalent methods and apparatuses within the scope of theinvention, in addition to those enumerated herein, will be apparent tothose skilled in the art from the foregoing descriptions. Suchmodifications and variations are intended to fall within the scope ofthe appended claims. The present invention is to be limited only by theterms of the appended claims, along with the full scope of equivalentsto which such claims are entitled. It is to be understood that thisinvention is not limited to particular methods, reagents, compoundscompositions or biological systems, which can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 cells refers to groupshaving 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers togroups having 1, 2, 3, 4, or 5 cells, and so forth.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. A method for treating or preventing an ophthalmiccondition in a mammalian subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of apeptide represented by the formula D-Arg-2′6′-Dmt-Lys-Phe-NH₂ orPhe-D-Arg-Phe-Lys-NH₂.
 2. The method of claim 1, wherein the ophthalmiccondition is selected from the group consisting of: diabeticretinopathy, cataracts, retinitis pigmentosa, glaucoma, maculardegeneration, choroidal neovascularization, retinal degeneration, andoxygen-induced retinopathy.
 3. The method of claim 1, wherein thepeptide is D-Arg-2′6′-Dmt-Lys-Phe-NH₂.
 4. The method of claim 1, whereinthe peptide is Phe-D-Arg-Phe-Lys-NH₂.
 5. The method of claim 1, whereinthe subject is a human.
 6. The method of claim 1, wherein the peptide isadministered intraocularly, iontophoretically, orally, topically,systemically, intravenously, subcutaneously, or intramuscularly.
 7. Themethod of claim 1 further comprising separately, sequentially, orsimultaneously administering a second active agent.
 8. The method ofclaim 7, wherein the second active agent is selected from the groupconsisting of: an antioxidant, a metal complexer, an anti-inflammatorydrug, an antibiotic, and an antihistamine.
 9. The method of claim 8,wherein the antioxidant is vitamin A, vitamin C, vitamin E, lycopene,selenium, α-lipoic acid, coenzyme Q, glutathione, or a carotenoid. 10.The method of claim 7, wherein the second active agent is selected fromthe group consisting of: aceclidine, acetazolamide, anecortave,apraclonidine, atropine, azapentacene, azelastine, bacitracin,befunolol, betamethasone, betaxolol, bimatoprost, brimonidine,brinzolamide, carbachol, carteolol, celecoxib, chloramphenicol,chlortetracycline, ciprofloxacin, cromoglycate, cromolyn,cyclopentolate, cyclosporin, dapiprazole, demecarium, dexamethasone,diclofenac, dichlorphenamide, dipivefrin, dorzolamide, echothiophate,emedastine, epinastine, epinephrine, erythromycin, ethoxzolamide,eucatropine, fludrocortisone, fluorometholone, flurbiprofen, fomivirsen,framycetin, ganciclovir, gatifloxacin, gentamycin, homatropine,hydrocortisone, idoxuridine, indomethacin, isoflurophate, ketorolac,ketotifen, latanoprost, levobetaxolol, levobunolol, levocabastine,levofloxacin, lodoxamide, loteprednol, medrysone, methazolamide,metipranolol, moxifloxacin, naphazoline, natamycin, nedocromil,neomycin, norfloxacin, ofloxacin, olopatadine, oxymetazoline,pemirolast, pegaptanib, phenylephrine, physostigmine, pilocarpine,pindolol, pirenoxine, polymyxin B, prednisolone, proparacaine,ranibizumab, rimexolone, scopolamine, sezolamide, squalamine,sulfacetamide, suprofen, tetracaine, tetracyclin, tetrahydrozoline,tetryzoline, timolol, tobramycin, travoprost, triamcinulone,trifluoromethazolamide, trifluridine, trimethoprim, tropicamide,unoprostone, vidarbine, xylometazoline, pharmaceutically acceptablesalts thereof, and combinations thereof.